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

Burn-induced red blood cell deformability and shape changes are modulated by sex hormones

After completing this article, readers should be able to: 1. Describe alterations of red blood cells due to bacterial toxins. 2. Describe physical alterations of neutrophils during activation. 3. Delineate the role of red and white blood cells in the impairment of microcirculation and organ damage in sepsis. 4. List drugs that may inhibit neutrophil activation and improve their deformability and their actions. Sepsis remains a major cause of morbidity and mortality in neonates, particularly among preterm infants. (1) Impaired microcirculatory blood flow plays a pivotal role in the development of clinical manifestations and organ dysfunctions in severe sepsis and septic shock. (2)(3)(4) If not corrected, microcirculatory dysfunction can progress to organ dysfunction and subsequently to organ failure and death. Restoration of microcirculatory dysfunction is, therefore, an important step in preventing long-term sequelae (including brain damage) and death of the patient. Both red and white blood cells must deform to pass through narrow channels whose diameters are less than those of the cells. (5) Impaired deformability of red and white blood cells may contribute to impaired microcirculatory blood flow in septicemia. The membranes of neonatal red blood cells (RBCs) deform more in response to a given force than adult cells and are, therefore, more flexible. (6)(7) On the other hand, neonatal RBCs are larger and require higher pressures to enter filter pores and micropipettes that have diameters below the resting cellular diameters. (8)(9) The larger volume and increased deformability of neonatal RBCs are responsible for another favorable property, the increased Fahraeus and Fahraeus-Lindqvist effect (ie, hematocrit and viscosity reduction when going from 500- to 50-mcm tubes). (10) These favorable flow properties of neonatal RBCs suggest that their increased cellular deformability is a prerequisite for adequate flow in arterioles and capillaries. We have shown that loss of …

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

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.

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.

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.

Trauma/hemorrhagic shock (T/HS) is known to cause changes in red blood cell (RBC) deformability and resting shape. Our previous studies have shown that proestrus female rats are more protected from shock-induced RBC damage than diestrus females or males. However, it is unclear whether female or male sex hormones can influence the severity of these alterations. Red blood cell deformability and shape were examined in proestrus female rats, and oophorectomized female rats, as well as in castrated and non-castrated male rats (5-10 animals per group) subjected to T/HS. Red blood cell deformability was measured by laser ektacytometry whereas erythrocyte shape was evaluated by scanning electron microscopy. Proestrus female rats subjected to T/HS did not show either significant RBC deformability changes (decrease in elongation index) or shape alterations (increase in the percentage of reversibly and irreversibly changed cells). Oophorectomized rats demonstrated more severe RBC changes than did non-oophorectomized rats. The degree of RBC damage was the same in castrated and non-castrated males, which was significantly worse than in proestrus females. Removal of female sex hormones increases the severity of T/HS-induced RBC changes, indicating that female sex hormones protect against RBC damage. In contrast, male sex hormones do not appear to modulate T/HS RBC dysfunction.

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.

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

This study introduces a novel method for evaluating red blood cell (RBC) deformability using a microfluidic platform equipped with constriction channels that mimic capillary vessels. The normalized transit velocity of RBCs at 50% compression ratio (V^ε = 0.5) is proposed as a quantitative index of RBC deformability. Experimental validation is conducted in two key phases. First, to validate the proposed index, a controlled reduction in RBC deformability is induced using varying concentrations of diamide. V^ε = 0.5 progressively decrease as the diamide concentration increased, confirming that the index tracks changes in RBC deformability. Second, to demonstrate the ability of the platform to detect disease-specific properties, RBCs from patients with iron deficiency anemia (IDA), thalassemia (Thal), and hereditary spherocytosis (HS) are analyzed. The results reveal elevated deformability from IDA and Thal patients, and reduced deformability from HS patients. High V^ε = 0.5 values consistently correlate with RBC characteristics known to enhance deformability-such as thin, elliptical morphology, a high surface-area-to-volume ratio, and low internal viscosity-whereas lower values are associated with characteristics that impair deformability. These findings highlight the impact of RBC characteristics on deformability and the importance of our microfluidic platform as a robust tool for investigating hematological diseases.

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

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.

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.

The main function of red blood cells (RBCs) is the transport of respiratory gases along the vascular tree. To fulfill their task, RBCs are able to elastically deform in response to mechanical forces and, pass through the narrow vessels of the microcirculation. Decreased RBC deformability was observed in pathological conditions linked to increased oxidative stress or decreased nitric oxide (NO) bioavailability, like hypertension. Treatments with oxidants and with NO were shown to affect RBC deformability ex vivo, but the mechanisms underpinning these effects are unknown. In this study we investigate whether changes in intracellular redox status/oxidative stress or nitrosation reactions induced by reactive oxygen species (ROS) or NO may affect RBC deformability. In a case-control study comparing RBCs from healthy and hypertensive participants, we found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls, while NO levels in RBCs were not significantly different. To study the effects of oxidants on RBC redox state and deformability, RBCs from healthy volunteers were treated with increasing concentrations of tert-butylhydroperoxide (t-BuOOH). We found that high concentrations of t-BuOOH (≥ 1 mM) significantly decreased the GSH/GSSG ratio in RBCs, decreased RBC deformability and increased blood bulk viscosity. Moreover, RBCs from Nrf2 knockout (KO) mice, a strain genetically deficient in a number of antioxidant/reducing enzymes, were more susceptible to t-BuOOH-induced impairment in RBC deformability as compared to wild type (WT) mice. To study the role of NO in RBC deformability we treated RBC suspensions from human volunteers with NO donors and nitrosothiols and analyzed deformability of RBCs from mice lacking the endothelial NO synthase (eNOS). We found that NO donors induced S-nitrosation of the cytoskeletal protein spectrin, but did not affect human RBC deformability or blood bulk viscosity; moreover, under unstressed conditions RBCs from eNOS KO mice showed fully preserved RBC deformability as compared to WT mice. Pre-treatment of human RBCs with nitrosothiols rescued t-BuOOH-mediated loss of RBC deformability. Taken together, these findings suggest that NO does not affect RBC deformability per se, but preserves RBC deformability in conditions of oxidative stress.

Red blood cells (RBCs) are the high specialized cells having the oxygen transport as the main function.They lost nucleus and mitochondria but they kept many elements of the molecular signaling pathways.Red blood cells alter their mechanical properties under realization of transport tasks -they can be deformed and unite each other (aggregations).There are some evidences that RBC mechanical changes happen under influence of signaling molecules, such as: receptors, ion channels, and enzymes.So, the aim of study was to investigate a role of receptors, ion channels, and enzymes in RBC micromechanical property alterations.For these purposes, RBCs were incubated with adrenoceptor agonists, inhibitors of enzymes and ion channels of the membrane, followed by registration of erythrocyte aggregation and deformability.It has been shown that adrenaline increased moderately RBC deformability, together with a marked rise in their aggregation (by 34 %), whereas alpha-1 agonist -phenylephrine did not markedly change RBC deformability, but strongly stimulated RBC aggregation, its increase was 53 %.On the contrary -beta-adrenoceptor agonist isoproterenol promoted with a marked increase in the deformability of red blood cells together with a moderate decrease in red blood cell aggregation.More significant and positive changes in RBC deformability were watched under the changing of enzyme activity.The average increase of the red cell deformability was 14 %.Stimulation of Ca 2+ entry into erythrocytes or blocking ion channels significantly altered the red blood cell deformability and their aggregation, especially under the influence of the Ca 2+ channel blocker -verapamil.The strongest effect of Ca 2+ was monitored for aggregation, it is changed on average by 44 %.The findings suggest that the mechanical properties of red blood cells and their transport capacity significantly changed under the effect of the activation or inhibition of the elements of molecular signaling pathways of cells (receptors, enzymes and ion channels).