Changes In Red Blood Cell Deformability Research Articles

Statin-treated RBC dynamics in a microfluidic porous-like network

The impact of therapeutic interventions on red blood cell (RBC) deformability and microscale transport is investigated, using statins as an exemplar. Human RBCs were treated in vitro with two commonly prescribed statins, atorvastatin and rosuvastatin, at clinically relevant concentrations. Changes in RBC deformability were quantified using a microfluidic-based ektacytometer and expressed in terms of the elongation index. Dilute suspensions of the statin-treated RBCs were then perfused through a microfluidic pillar array, at a constant flow rate and negligible inertia, and imaged. Particle Tracking Velocimetry (PTV) was applied to track RBCs, identify preferential paths and estimate their velocities, whereas image processing was used to estimate cell dynamics, perfusion metrics and distributions. The findings were compared against those of healthy, untreated cells. Statins enhanced RBC deformability in agreement with literature. The extent of enhancement was found to be statin-dependent. The softer statin-treated cells were found to flow in straight, less tortuous paths, spend more time inside the pillar array and exhibit lower velocities compared to healthy RBCs, attributed to their enhanced deformation and longer shape recovery time upon impact with the array posts. The in vitro microfluidic approach demonstrated here may serve as a monitoring tool to personalise and maximise the outcome of a therapeutic treatment.

Pathophysiology of Red Blood Cell Dysfunction in Diabetes and Its Complications.

Diabetes Mellitus (DM) is a complex metabolic disorder associated with multiple microvascular complications leading to nephropathy, retinopathy, and neuropathy. Mounting evidence suggests that red blood cell (RBC) alterations are both a cause and consequence of disturbances related to DM-associated complications. Importantly, a significant proportion of DM patients develop varying degrees of anemia of confounding etiology, leading to increased morbidity. In chronic hyperglycemia, RBCs display morphological, enzymatic, and biophysical changes, which in turn prime them for swift phagocytic clearance from circulation. A multitude of endogenous factors, such as oxidative and dicarbonyl stress, uremic toxins, extracellular hypertonicity, sorbitol accumulation, and deranged nitric oxide metabolism, have been implicated in pathological RBC changes in DM. This review collates clinical laboratory findings of changes in hematology indices in DM patients and discusses recent reports on the putative mechanisms underpinning shortened RBC survival and disturbed cell membrane architecture within the diabetic milieu. Specifically, RBC cell death signaling, RBC metabolism, procoagulant RBC phenotype, RBC-triggered endothelial cell dysfunction, and changes in RBC deformability and aggregation in the context of DM are discussed. Understanding the mechanisms of RBC alterations in DM provides valuable insights into the clinical significance of the crosstalk between RBCs and microangiopathy in DM.

The Pyruvate Kinase Activator Mitapivat Improves Red Blood Cell Deformability and Sickling Kinetics in Adult Patients with Sickle Cell Disease

Background: A hallmark of sickle cell disease (SCD) is compromised red blood cell (RBC) deformability. Polymerization of hemoglobin S (HbS) underlies this "sickling" which results in altered biomechanical properties that impact rheology and hemodynamics (Barabino et al, 2010). There have been exciting clinical developments in anti-sickling therapies - including enhancement of red cell pyruvate kinase (PKR) modality. Importantly, deformability measurements of RBCs via ektacytometry are increasingly used to aid the assessment of their therapeutic efficacy. Here we employed the Laser assisted Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics), which permits the evaluation of RBC deformability as a function of shear stress, osmotic pressure, and oxygen tension, to evaluate the changes in RBC deformability in subjects undergoing treatment with mitapivat (AG-348), a PKR activator under clinical development for sickle cell disease. We analyzed changes in deformability measurements of RBCs in HbSS subjects after 4 weeks and 12 weeks of mitapivat therapy and compared them to t50 - a single parameter measure of sickling kinetics (Dunkelberger et al, 2018). Methods: We studied subjects currently enrolled under IRB approved clinical trial NCT04610866 that evaluates the long-term safety and tolerability of mitapivat in SCD patients. Fifteen subjects (aged 25 - 57 years; 5 females, 10 males; African/African-American) were evaluated, though not all endpoints were available for every patient across all assays. Fresh whole blood (collected in EDTA tubes) was obtained at baseline (prior to drug initiation), after 4 weeks and after 12 weeks of therapy. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing same-day LORRCA assays. The Elongation Index (EI) is a measure of the cells' ability to undergo deformation at different physiologically relevant conditions. EI was assessed at increasing shear stresses of .3 Pascals (Pa), .95 Pa, 3 Pa, 9.49 Pa, and 30 Pa (elongation assay). Parameters derived under continuous osmotic changes (0-600mOsm/kg - osmoscan) include O min which informs on RBC fragility, O Hyper which informs on hydration state and corresponds to 50% of the maximum deformability in the hypertonic region, EI max (osmo) which reveals maximum deformability and informs on RBC cytoskeletal mechanics, and O Max which gives the osmolality at which EI Max is achieved. Point of Sickling (POS) gives the oxygen tension at which the cells start to sickle during nitrogen facilitated deoxygenation of the sample (oxyscan). These parameters of deformability are generated by LORRCA software. For all assays, we analyzed the % change from baseline after 4 weeks and 12 weeks of therapy (Wilcoxon signed rank test, R v1.4.1106) and compared these changes to those in t50 (Spearman correlation test, Prism v9) (Figure 1). Results: Mitapivat therapy resulted in increased elongation indices across all shear stress levels, with significance reached at shears of .3 Pa, .95 Pa, and 3 Pa (respective mean % changes of 78.5, 181.8, and 23.0 (4 weeks) and 101.2, 212.9, and 30.1 (12 weeks)) suggesting improved deformability of RBCs. Likewise, significant increases in O hyper (mean % changes of 2.8 and 3.4) and osmo EI Max (mean % changes 7.9 and 9.5) respectively, indicate improvements in RBC hydration and cytoskeletal dynamics in response to changing osmotic pressures. Furthermore, a significant decrease in the average point of sickling was noted at both 4 weeks and 12 weeks (-20.15% and -16.8%, respectively), suggesting RBCs were susceptible to sickling at partial pressures of oxygen lower than those observed before therapy. Conclusion: Altogether, these findings indicate that mitapivat therapy results in improved ability of RBCs to withstand deformational changes in response to shear stress, osmotic pressure, and decreasing oxygen tension, likely via bolstered membrane integrity. Interestingly, when compared to t50 values, significant correlation was observed at all levels of shear stress and at the maximal deformability (EI max) achieved during both the osmoscan and oxyscan assays. Acknowledgement: This study is part of a Cooperative Research and Development Agreement (CRADA) with AGIOS Pharmaceuticals, Inc., Cambridge, MA, United States Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Shear-Stress-Gradient and Oxygen-Gradient Ektacytometry in Sickle Cell Patients at Steady State and during Vaso-Occlusive Crises.

Oxygen gradient ektacytometry (oxygenscan) measures the changes in red blood cell (RBC) deformability in normoxia and during deoxygenation. We investigated the changes in RBC deformability, measured by both oxygenscan and classical shear-stress-gradient ektacytometry, in 10 patients with sickle cell disease (SCD) during vaso-occlusive crisis (VOC) versus steady state. Oxygenscan and shear-stress-gradient ektacytometry parameters were also measured in 38 SCD patients at steady state on two different occasions. Shear-stress-gradient ektacytometry parameters, maximal RBC deformability at normoxia and the minimum RBC deformability during deoxygenation were lower during VOC compared to steady state. The oxygen partial pressure at which RBCs started to sickle (PoS) was not significantly affected by VOC, but the results were very heterogeneous: the PoS increased in 5 in 10 patients and decreased in 4 in 10 patients. Both oxygenscan and shear-stress-gradient ektacytometry parameters remained unchanged in patients at steady state between two sets of measurements, performed at 17 ± 8 months intervals. In conclusion, the present study showed that both oxygen gradient ektacytometry and shear-stress-gradient ektacytometry are sensitive to disease activity in SCD, and that both techniques give comparable results; however, the oxygen-dependent propensity of RBCs to sickle was highly variable during VOC.

Reduced deformability contributes to impaired deoxygenation-induced ATP release from red blood cells of older adult humans.

Red blood cells (RBCs) release ATP in response to deoxygenation, which can increase blood flow to help match oxygen supply with tissue metabolic demand. This release of ATP is impaired in RBCs from older adults, but the underlying mechanisms are unknown. In this study, improving RBC deformability in older adults restored deoxygenation-induced ATP release, whereas decreasing RBC deformability in young adults reduced ATP release to the level of that of older adults. In contrast, treating RBCs with a phosphodiesterase 3 inhibitor did not affect ATP release in either age group, possibly due to intact intracellular signalling downstream of deoxygenation as indicated by preserved cAMP and ATP release responses to pharmacological Gi protein activation in RBCs from older adults. These findings are the first to demonstrate that the age-related decrease in RBC deformability is a primary mechanism of impaired deoxygenation-induced ATP release, which may have implications for treating impaired vascular control with advancing age. In response to haemoglobin deoxygenation, red blood cells (RBCs) release ATP, which binds to endothelial purinergic receptors and stimulates vasodilatation. This ATP release is impaired in RBCs from older vs. young adults, but the underlying mechanisms are unknown. Using isolated RBCs from young (24±1years) and older (65±2years) adults, we tested the hypothesis that age-related changes in RBC deformability (Study 1) and cAMP signalling (Study 2) contribute to the impairment. RBC ATP release during normoxia ( ∼112mmHg) and hypoxia ( ∼20mmHg) was quantified with the luciferin-luciferase technique following RBC incubation with Y-27632 (Rho-kinase inhibitor to increase deformability), diamide (cell-stiffening agent), cilostazol (phosphodiesterase 3 inhibitor), or vehicle control. The mean change in RBC ATP release from normoxia to hypoxia in control conditions was significantly impaired in older vs. young (∼50% vs. ∼120%; P<0.05). RBC deformability was also lower in older vs. young as indicated by a higher RBC transit time (RCTT) measured by blood filtrometry (RCTT: 8.541±0.050 vs. 8.234±0.098a.u., respectively; P<0.05). Y-27632 improved RBC deformability (RCTT: 8.228±0.083) and ATP release (111.7±17.2%) in older and diamide decreased RBC deformability (RCTT: 8.955±0.114) and ATP release (67.4±11.8%) in young (P<0.05), abolishing the age group differences (P>0.05). Cilostazol did not change ATP release in either age group (P>0.05), and RBC cAMP and ATP release to pharmacological Gi protein activation was similar in both groups (P>0.05). We conclude that decreased RBC deformability is a primary contributor to age-related impairments in RBC ATP release, which may have implications for impaired vascular control with advancing age.

Deformation of Red Blood Cells, Air Bubbles, and Droplets in Microfluidic Devices: Flow Visualizations and Measurements.

Techniques, such as micropipette aspiration and optical tweezers, are widely used to measure cell mechanical properties, but are generally labor-intensive and time-consuming, typically involving a difficult process of manipulation. In the past two decades, a large number of microfluidic devices have been developed due to the advantages they offer over other techniques, including transparency for direct optical access, lower cost, reduced space and labor, precise control, and easy manipulation of a small volume of blood samples. This review presents recent advances in the development of microfluidic devices to evaluate the mechanical response of individual red blood cells (RBCs) and microbubbles flowing in constriction microchannels. Visualizations and measurements of the deformation of RBCs flowing through hyperbolic, smooth, and sudden-contraction microchannels were evaluated and compared. In particular, we show the potential of using hyperbolic-shaped microchannels to precisely control and assess small changes in RBC deformability in both physiological and pathological situations. Moreover, deformations of air microbubbles and droplets flowing through a microfluidic constriction were also compared with RBCs deformability.

Repetitive Supra-Physiological Shear Stress Impairs Red Blood Cell Deformability and Induces Hemolysis.

The supra-physiological shear stress that blood is exposed to while traversing mechanical circulatory assist devices affects the physical properties of red blood cells (RBCs), impairs RBC deformability, and may induce hemolysis. Previous studies exploring RBC damage following exposure to supra-physiological shear stress have employed durations exceeding clinical instrumentation, thus we explored changes in RBC deformability following exposure to shear stress below the reported "hemolytic threshold" using shear exposure durations per minute (i.e., duty-cycles) reflective of that employed by circulatory assist devices. Blood collected from 20 male donors, aged 18-38 years, was suspended in a viscous medium and exposed to an intermittent shear stress protocol of 1 s at 100 Pa, every 60 s for 60 duty-cycles. During the remaining 59 s/min, the cells were left at stasis until the subsequent duty-cycle commenced. At discrete time points (15/30/45/60 duty-cycles), an ektacytometer measured RBC deformability immediately after shear exposure at 100 Pa. Plasma-free hemoglobin, a measurement of hemolysis, was quantified via spectrophotometry. Supra-physiological shear stress impaired RBC properties, as indicated by: (1) decreased maximal elongation of RBCs at infinite shear stress following 15 duty-cycles (P <0.05); (2) increased real-time RBC deformability during application of the supra-physiological shear stress protocol (100 Pa) following exposure to 1 duty-cycle (F (1.891, 32.15) = 12.21, P = 0.0001); and (3) increased plasma-free hemoglobin following 60 duty-cycles (P < 0.01). The present study indicates that exposure of RBCs to short-term, repeated supra-physiological shear stress, impairs RBC deformability, with the extent of impairment exacerbated with each duty-cycle, and ultimately precipitates hemolysis.

Microfluidic analysis of red blood cell deformability as a means to assess hemin-induced oxidative stress resulting from Plasmodium falciparum intraerythrocytic parasitism.

Hemolytic anemia is one of the hallmarks of malaria and leads to an increase in oxidized heme (hemin) within the plasma of infected individuals. While scavenger proteins sequester much of the circulating heme, it has been hypothesized that extracellular heme may play a central role in malaria pathogenesis. We have previously developed the multiplex fluidic plunger (MFP) device for the measurement of red blood cell (RBC) deformability. Here, we demonstrate that the measurement of changes in RBC deformability is a sensitive method for inferring heme-induced oxidative stress. We further show that extracellular hemin concentration correlates closely with changes in RBC deformability and we confirm that this biophysical change correlates with other indicators of cell stress. Finally, we show that reduced erythrocyte deformability corresponds with both erythrophagocytosis and RBC osmotic fragility. The MFP microfluidic device presents a simple and potentially inexpensive alternative to existing methods for measuring hemolytic cell stress that could ultimately be used to perform clinical assessment of disease progression in severe malaria.

326 - The Role of Cytoskeletal S-Nitrosation in Red Blood Cell Deformability

The deformability of red blood cells (RBC) is an important characteristic of RBC functionality, and contributes to define blood rheological properties and blood viscosity. The ability of RBCs to change their shape mainly depends on the flexibility of the interactions between cytoskeletal proteins, in particular of the structure of the main cysteine rich protein spectrin. In this study we aimed to analyze the effects of S-nitrosation of spectrin on changes in RBC deformability. We found that treatment with S-nitrosocystein (SNOC) leads to S-nitrosation of purified spectrin as assessed by chemiluminescence detection (CLD). Similarly, SNOC-induced nitrosation of cytoskeletal proteins of RBC ghosts and RBC were measured by CLD and the biotin switch assay. Western blotting following the biotin switch assay confirmed that spectrin was one of the nitrosated cytoskeletal proteins in RBC ghosts and RBCs. However, treatment with SNOC or DEA/NO did not affect deformability of RBCs as assessed by ektacytometry measured in a LORRCA system, nor blood viscosity measured in a low shear rotational viscometer under the same shear rate conditions. Instead, we found that changes in redox state of RBC by treatment with high concentrations of tert-Butyl hydroperoxide decreased deformability and increased blood viscosity. Our results demonstrate that incubation of RBC, RBC ghosts and purified spectrin with a nitrosothiol result in significantly increased S-nitrosation of RBC cytoskeletal spectrin. However, nitrosation did not improve RBC deformability or change blood viscosity. Hence, in our setting change in redox state/oxidative modification rather than nitrosation reactions of cytoskeletal proteins like spectrin have an impact on RBC deformability and blood viscosity.

Two-dimensional numerical simulation of the red blood cell floating in a plasma-alcohol solution through stenosis in a microvessel

A two-dimensional computational model of a single red blood cell (RBC) floating in a plasma-alcohol solution through a microchannel with stenosis was created using the Arbitrary Lagrangian-Eulerian (ALE) method with moving mesh for a fluid structure interaction problem. Cell deformability and stability were studied in a plasma-alcohol solution at different fluid flow conditions during movement through the channel with stenosis. Different results were obtained for different input parameters. Motion through 45% and 70% stenoses with the high and law velocities of the RBC and different viscosities was analyzed and successfully simulated. Results show that changes in RBC deformability were due to the effects of alcohol. Changes in behavior during motion were also observed. At low shear rate and high surrounding fluid viscosity the RBC showed a tendency to rotate during movement. The proposed model with its coupling of structural and fluid analysis techniques could be useful to understand the effect of alcohol on the RBC passing through stenosis.

Human red blood cell behavior under homogeneous extensional flow in a hyperbolic-shaped microchannel

It is well known that certain pathological conditions result in a decrease of red blood cells (RBCs) deformability and subsequently can significantly alter the blood flow in microcirculation, which may block capillaries and cause ischemia in the tissues. Microfluidic systems able to obtain reliable quantitative measurements of RBC deformability hold the key to understand and diagnose RBC related diseases. In this work, a microfluidic system composed of a microchannel with a hyperbolic-shaped contraction followed by a sudden expansion is presented. We provide a detailed quantitative description of the degree of deformation of human RBCs under a controlled homogeneous extensional flow field. We measured the deformation index (DI) as well as the velocity of the RBCs travelling along the centerline of the channel for four different flow rates and analyze the impact of the particle Reynolds number. The results show that human RBC deformation tends to reach a plateau value in the region of constant extensional rate, the value of which depends on the extension rate. Additionally, we observe that the presence of a sudden expansion downstream of the hyperbolic contraction modifies the spatial distribution of cells and substantially increases the cell free layer (CFL) downstream of the expansion plane similarly to what is seen in other expansion flows. Beyond a certain value of flow rate, there is only a weak effect of inlet flow rates on the enhancement of the downstream CFL. These in vitro experiments show the potential of using microfluidic systems with hyperbolic-shaped microchannels both for the separation of the RBCs from plasma and to assess changes in RBC deformability in physiological and pathological situations for clinical purposes. However, the selection of the geometry and the identification of the most suitable region to evaluate the changes on the RBC deformability under extensional flows are crucial if microfluidics is to be used as an in vitro clinical methodology to detect circulatory diseases.

P70: Molecular and physiological aspects connecting red cell eNOS, red blood cell deformability and decreased microvascular function in patients with essential hypertension

Background Arterial hypertension (HT) is a complex multifactorial condition associated with cardiovascular disease. Independent studies show that HT is associated with microcirculatory and hemorheological alterations, such as decreased red blood cell (RBC) deformability, as well as decreased endothelial function and nitric oxide (NO) bioavailability. Recently we found RBC carry an active endothelial nitric oxide synthase (eNOS), which is decreased in cardiovascular disease.Hypothesis: We hypothesized that a reduced RBC deformability may contribute to induce a hypertensive phenotype via impairment of microcirculatory conductance. Methods To evaluate hemorehological alterations in HT, patients ( n = 10) with HT and age-and risk factors-matched subjects ( n = 10) without HT (control) were recruited. High BP was defined as systolic BP > 135 mm Hg and diastolic BP > 85 mm in 24 h BP measurements. RBC deformability was measured by laser-assisted optical rotational cell analyzer (LORCA) and assessed as elongation index (EI) at 1.73 Pa. RBC redox state (ROS, GSH) was assessed by labeling with diclorodihydrofluorescein and thiol-tracker and flow cytometry. Cutaneous microvascular function as post-occlusion reactive hyperemia was measured by noninvasive laser Doppler Perfusion imaging and quantified as area under the curve (AUC). Endothelial function of the brachial artery was assessed as flow-mediated dilatation (FMD) by high resolution ultrasound. Deformability of RBC from eNOS −/− mice was evaluated and compared to wild type (WT) C57Bl/6 mice. To evaluate NO and NOS-dependent changes in RBC deformability, RBC from healthy individuals ( n = 6) were treated with NO donors, NOS inhibitors and guanylate cyclase inhibitors. Measurements of cGMP production in RBC were assessed by ELISA. A stepwise linear regression as well as a multivariate linear regression model were performed to identify independent predictors for systolic BP. Results In the HT group EI (0.300 ± 0.007 vs. 0.326 ± 0.007, p = 0.013), AUC (1657 ± 422 vs. 2313 ± 473 PU; p = 0.009). and FMD (6.2% ± 0.7% vs. 5.0% ± 0.3 % p = 0.010) was reduced in the HT group, suggesting decreased deformability, shorter lasting vasodilation of microvessels and endothelial dysfunction. In RBC from HT patients the ROS levels increased and the GSH levels decreased, indicating increased oxidative stress. Univariate inverse correlations were found between systolic BP and AUC ( r = −0.485; p r = 0.726; p R 2 = 0.607, p = 0.004). In a translational approach we analyzed deformability in eNOS −/− mice, characterized by a dysfunctional endothelium and hypertension. eNOS −/− RBC are less deformable than WT RBC, show increased ROS and decreased GSH, similarly to the HT grouP Moreover, NO donors increased EI of RBC from healthy individuals, while NOS inhibition as well as guanlyte cyclase inhibition decreased cGMP levels and EI indicating that NO/NOS/cGMP dependent pathways control deformability in RBC. Conclusion The NO/NOS/cGMP pathway controls RBC mechanoproperties, and RBC deformability is an independent predictor of increased blood pressure in patients with arterial hypertension. Disclosure Nothing to disclose.

Shear stress-induced improvement of red blood cell deformability

Classically, it is known that red blood cell (RBC) deformability is determined by the geometric and material properties of these cells. Experimental evidence accumulated during the last decade has introduced the concept of active regulation of RBC deformability. This regulation is mainly related to altered associations between membrane skeletal proteins and integral proteins, with the latter serving to anchor the skeleton to the lipid matrix. It has been hypothesized that shear stress induces alterations of RBC deformability: the current study investigated the dynamics of the transient improvement in deformability induced by shear stress at physiologically-relevant levels. RBC were exposed to various levels of shear stress (SS) in a Couette type shearing system that is part of an ektacytometer, thus permitting the changes in RBC deformability during the application of SS to be monitored. Initial studies showed that there is an increase in deformability of the RBC subjected to SS in the range of 5-20 Pa, with kinetics characterized by time constants of a few seconds. Such improvement in deformability, expressed by an elongation index (EI), was faster with higher levels of SS and hence yielded shorter time constants: absolute values of EI increased by 3-8% of the starting level. Upon the removal of the shear stress, this response by RBC was reversible with a slower time course compared to the increase in EI during application of SS. Increased calcium concentration in the RBC suspending medium prevented the improvement of deformability. It is suggested that the improvement of RBC deformability by shear forces may have significant effects on blood flow dynamics, at least in tissues supplied by blood vessels with impaired vasomotor reserve, and may therefore serve as a compensating mechanism for the maintenance of adequate microcirculatory perfusion.

Role molecular signaling pathways in changes of red blood cell deformability

This study was designed to investigate the dependency of the red blood cell deformability upon activation of extra- and intracellular signaling pathways. Exposures of red blood cells (RBCs) to catecholamines and to insulin led to positive change in the RBC deformability. When forskolin, a stimulator of adenylyl cyclase (AC), was added to RBC suspension, the RBC deformability was increased. Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. The inhibitors of phosphodiesterase (PDE) activity increased red cell deformability. These results revealed a considerable role of the AC-cAMP signaling system in the regulation of red blood cell deformability. The rise of the red blood cell Ca(2+) influx, stimulated by mechanical loading or A23187 was accompanied by a marked lowering of RBC deformability. At the same time blocking of Ca(2+) entry into RBC by verapamil or Ca(2+) chelating by EGTA led to significant deformability rise. The comparison of the effect of the different protein kinases on the red blood cell deformability showed that it was altered more considerable under PKA activation by forskolin or dB-cAMP than by other protein kinases. There was a lesser but quite statistically significant effect of tyrosine protein kinase (TPK) on RBC microrheology. Whereas the microrheological effect of PKC was not so considerable. The problem of the short-term regulation of red blood cell microrheology is examined. The latter includes: the modes of activation of extra- and intracellular molecular signaling pathways, ligand - receptor interaction, second messengers, membrane protein phosphorylation. On the whole the total data clearly show that the red cell deformability changes are connected with activation of different extra - and intracellular signaling pathways. It seems reasonable to suppose that red blood cell deformability changes were mainly associated with activation of the AC-cAMP-PKA pathway, and with decrease of Ca(2+) entry into cells.

Eadie-Hofstee analysis of red blood cell deformability

In recent years, linear data transformation has become an accepted method to simplify the analysis of red blood cell (RBC) deformability curves obtained by ektacytometry. In this study, we introduce the Eadie-Hofstee transformation as an alternative linearization method for the analysis of RBC deformability. RBCs were treated with hydrogen peroxide (H(2)O(2)), tert-butyl-hydroperoxide (t-BuOOH), or methyl β-cyclodextrin (MβCD) and analyzed via ektacytometry (LORCA). RBC hemopathological clinical isolates (hereditary spherocytosis and α-thalassemia) were also analyzed by LORCA. Following ektacytometry, Eadie-Hofstee linearization was performed to obtain the maximum deformability (EI(max)) and shear stress at half maximal deformation (K(EI)) parameters. Significant changes in deformability parameters were observed with all agents tested. For H(2)O(2) and t-BuOOH, the K(EI) values increased significantly accompanied by marginal changes in EI(max), while treatment with MβCD resulted in a dose dependant decrease in EI(max). Contrasting deformability profiles were also observed in the two hematological disorders tested. In this study we have demonstrated the ability of Eadie-Hofstee linearization to detect and resolve changes in RBC deformability induced in vitro as well as deformability changes associated with in vivo hematological disorders. This technique shows promise in basic research, blood bank and clinical hematology settings.

A lab-on-a-chip capillary network for red blood cell hydrodynamics

The main function of red blood cells (RBCs) is to circulate oxygen and carbon dioxide throughout the human body. Accurate modeling of the transportation mechanism of RBCs inside microvessels will lead to better clinical diagnosis and prophylaxis of blood disease. This study combined hydrodynamics and basic circuit theory to model the fluid mechanisms of the circulation of blood cells inside capillaries. The variations of physical properties inside the capillaries due to clogging by RBCs were analyzed. A lab-on-a-chip for RBC deformability diagnosis was fabricated using soft lithography. Real experiments were conducted to verify the theoretical analysis and illustrated the capability of the device which was able to observe pathological changes in RBC deformability. The proposed device could be a convenient tool in the field of blood rheology and clinical applications.

Crosstalk between adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism under red blood cell microrheological changes

There are evidences that red blood cell (RBC) deformation and aggregation change under their incubation with catecholamines and it is connected with activation of intracellular signaling pathways. The present study was designed to explore the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism of RBCs together with their microrheological changes. The washed RBCs were resuspended in PBS. In each of the three research sessions RBC suspensions were divided into two aliquots: 1) control (without drug) and 2) with an appropriate drug. After cell incubation RBC deformability (RBCD) and aggregation (RBCA) were estimated. RBC incubation with catecholamines resulted in RBCD changes by 18-30%. RBCs incubation with forskolin facilitated an increase of RBCD by 17% (p < 0.05). A significant deformability rise under dB-AMP incubation was found by 27% (p < 0.01). Ca2+ cell influx, stimulated by A23187, was accompanied by an increase of RBCA; whereas red cell deformability was changed only slightly. On the other hand, Ca2+ entry blocking into the cells by verapamil has led to significant RBCA decrease and RBCD rise. The obtained results make us believe that RBCD change was closely associated with Ca2+ control mechanisms. An effect of Ca2+ concentration increase on RBC microrheology was removed, if it was preliminary added to incubation medium EGTA as Ca2+ chelator. It was found that all four PDE inhibitors: IBMX, vinpocetine, rolipram, pentoxifylline decreased RBCA significantly and, quite the contrary, they increased red cell deformability. Our data have shown that Ca2+ entry increase was accompanied by red cell aggregation rise, while adenylyl cyclase-cAMP system stimulation led to red cell deformability increase and its aggregation lowered. The crosstalk between two intracellular signaling systems is probably connected with phosphodiesterase activity.

Susceptibility of oxidative stress on red blood cells exposed to gamma rays: Hemorheological evaluation

Irradiation has been shown to induce biochemical changes in stored red blood cells (RBCs) and to generate reactive oxygen species (ROS). This study evaluated the hemorheological properties, the degree of lipid peroxidation and the oxidative susceptibility of irradiated RBCs. Furthermore, we investigated the radioprotective role of N-t-butyl hydroxylamine (NtBHA) against gamma-ray exposure of RBCs. RBC concentrates were irradiated with a minimum dose of 25 Gy, and were exposed to FeSO4 to examine the oxidative susceptibility. RBC deformability was evaluated by the use of a microfluidic ektacytometer, in relation to the hematological and biochemical properties. The deformability of the irradiated RBCs was significantly lower than that of control. Exposure to gamma rays significantly increased the mean corpuscular volume (MCV) and lipid peroxidation. Changes in RBC deformability were more prominent in irradiated RBCs than in non-irradiated RBCs also under conditions of oxidative stress. The deformability of NtBHA treated RBCs prior to irradiation was not altered as compared with irradiated RBCs not treated with NtBHA. In conclusion, irradiation reduces RBC deformability during storage and the irradiated RBCs seem susceptible to oxidative stress. NtBHA may not have a protective role against the effects of gamma-ray exposure in RBCs but further evaluation of NtBHA or another radioprotective compound is required.

Viscoelasticity of the human red blood cell

Because the red blood cell (RBC) is often larger than capillary diameter, an important determinant of the microcirculation is RBC deformability. Compromised deformability of the RBC is thought to play a role in circulatory pathologies associated with diabetes mellitus and malaria. Here we report the complex modulus of the single isolated RBC measured over wide ranges of forcing frequency and amplitude. Healthy RBCs were obtained by fingerprick, washed with phosphate-buffered saline, and allowed to adhere on poly-L-lysine coated glass substrate at room temperature. Functionalized ferromagnetic microbeads (2.3 μm dia.) were attached to the RBC membrane and then subjected to an oscillatory magnetic field. The resulting torque caused the cell to deform. From the oscillatory forcing and resulting bead motions tracked optically, we computed elastic and frictional moduli, g’ and g” in 24 cells from 0.1 to 100 Hz. g’ was nearly frequency independent and dominated the response at all but the highest frequencies measured. However, g” increased with frequency as a power law with an exponent of 0.64, a result not predicted by any simple model. A linear range of amplitude response was observed, but nonlinearities became apparent as forcing amplitude increased. While mechanism accounting for such dynamics of RBC deformation remains unclear, methods described here leave us well positioned to investigate how RBC deformability changes with temperature or malarial infection. Supported by HL33009/65960/59682

Mesenteric lymph duct ligation decreases red blood cell alterations caused by acute pancreatitis

Background Both experimental and clinical studies have shown that acute pancreatitis (AP) causes a significant decrease in red blood cell (RBC) deformability. The mechanisms by which AP induces RBC injury are unknown. The purpose of this study was to test the hypothesis that factors carried in the mesenteric lymph after an attack of AP significantly contribute to the RBC injury observed in AP. Methods RBC deformability was determined by means of laser-assisted ektacytometry in mesenteric lymph duct–ligated and non-ligated rats subjected to AP and in sham-operated animals. Results AP was associated with significant alterations of RBC deformability indices, namely the elongation index and half maximal RBC elongation. Pancreatitis-induced RBC deformability changes were partially prevented by mesenteric lymph duct ligation. Conclusions Mesenteric lymph in AP contains factors that cause RBC damage, which is manifested by decreased deformability. Interruption of the lymph flow from the injured gut into the bloodstream decreases these RBC alterations.