A framework of computer vision-enhanced microfluidic approach for automated assessment of the transient sickling kinetics in sickle red blood cells.

The occurrence of vaso-occlusive crisis greatly depends on the competition between the sickling delay time and the transit time of individual sickle cells, i.e., red blood cells (RBCs) from sickle cell disease (SCD) patients, while they are traversing the circulatory system. Many drugs for treating SCD work by inhibiting the polymerization of sickle hemoglobin (HbS), effectively delaying the sickling process in sickle cells (SS RBCs). Most previous studies on screening anti-sickling drugs, such as voxelotor, rely on in vitro testing of sickling characteristics, often conducted under prolonged deoxygenation for up to 1 hour. However, since the microcirculation of RBCs typically takes less than 1 minute, the results of these studies may be less accurate and less relevant for in vitro-in vivo correlation. In our current study, we introduce a computer vision-enhanced microfluidic framework designed to automatically capture the transient sickling kinetics of SS RBCs within a 1-min timeframe. Our study has successfully detected differences in the transient sickling kinetics between vehicle control and voxelotor-treated SS RBCs. This approach has the potential for broader applications in screening anti-sickling therapies.

Signaling-biophysical modeling unravels mechanistic control of red blood cell phagocytosis by macrophages in sickle cell disease.

Red blood cell (RBC) aging manifests through progressive changes in cell morphology, rigidity, and expression of membrane proteins. To maintain the quality of circulating blood, splenic macrophages detect the biochemical signals and biophysical changes of RBCs and selectively clear them through erythrophagocytosis. In sickle cell disease (SCD), RBCs display alterations affecting their interaction with macrophages, leading to aberrant phagocytosis that may cause life-threatening spleen sequestration crises. To illuminate the mechanistic control of RBC engulfment by macrophages in SCD, we integrate a system biology model of RBC-macrophage signaling interactions with a biophysical model of macrophage engulfment, as well as in vitro phagocytosis experiments using the spleen-on-a-chip technology. Our modeling framework accurately predicts the phagocytosis dynamics of RBCs under different disease conditions, reveals patterns distinguishing normal and sickle RBCs, and identifies molecular targets including Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP1) and cluster of differentiation 47 (CD47)/signal regulatory protein α (SIRPα) as therapeutic targets to facilitate the controlled clearance of sickle RBCs in the spleen.

A combined computational and experimental investigation of the filtration function of splenic macrophages in sickle cell disease.

Being the largest lymphatic organ in the body, the spleen also constantly controls the quality of red blood cells (RBCs) in circulation through its two major filtration components, namely interendothelial slits (IES) and red pulp macrophages. In contrast to the extensive studies in understanding the filtration function of IES, fewer works investigate how the splenic macrophages retain the aged and diseased RBCs, i.e., RBCs in sickle cell disease (SCD). Herein, we perform a computational study informed by companion experiments to quantify the dynamics of RBCs captured and retained by the macrophages. We first calibrate the parameters in the computational model based on microfluidic experimental measurements for sickle RBCs under normoxia and hypoxia, as those parameters are not available in the literature. Next, we quantify the impact of key factors expected to dictate the RBC retention by the macrophages in the spleen, namely, blood flow conditions, RBC aggregation, hematocrit, RBC morphology, and oxygen levels. Our simulation results show that hypoxic conditions could enhance the adhesion between the sickle RBCs and macrophages. This, in turn, increases the retention of RBCs by as much as four-fold, which could be a possible cause of RBC congestion in the spleen of patients with SCD. Our study on the impact of RBC aggregation illustrates a 'clustering effect', where multiple RBCs in one aggregate can make contact and adhere to the macrophages, leading to a higher retention rate than that resulting from RBC-macrophage pair interactions. Our simulations of sickle RBCs flowing past macrophages for a range of blood flow velocities indicate that the increased blood velocity could quickly attenuate the function of the red pulp macrophages on detaining aged or diseased RBCs, thereby providing a possible rationale for the slow blood flow in the open circulation of the spleen. Furthermore, we quantify the impact of RBC morphology on their tendency to be retained by the macrophages. We find that the sickle and granular-shaped RBCs are more likely to be filtered by macrophages in the spleen. This finding is consistent with the observation of low percentages of these two forms of sickle RBCs in the blood smear of SCD patients. Taken together, our experimental and simulation results aid in our quantitative understanding of the function of splenic macrophages in retaining the diseased RBCs and provide an opportunity to combine such knowledge with the current knowledge of the interaction between IES and traversing RBCs to apprehend the complete filtration function of the spleen in SCD.

A combined computational and experimental investigation of the filtration function of splenic macrophages in sickle cell disease.

Being the largest lymphatic organ in the body, the spleen also constantly controls the quality of red blood cells (RBCs) in circulation through its two major filtration components, namely interendothelial slits (IES) and red pulp macrophages. In contrast to the extensive studies in understanding the filtration function of IES, there are relatively fewer works on investigating how the splenic macrophages retain the aged and diseased RBCs, i.e., RBCs in sickle cell disease (SCD). Herein, we perform a computational study informed by companion experiments to quantify the dynamics of RBCs captured and retained by the macrophages. We first calibrate the parameters in the computational model based on microfluidic experimental measurements for sickle RBCs under normoxia and hypoxia, as those parameters are not available in the literature. Next, we quantify the impact of a set of key factors that are expected to dictate the RBC retention by the macrophages in the spleen, namely, blood flow conditions, RBC aggregation, hematocrit, RBC morphology, and oxygen levels. Our simulation results show that hypoxic conditions could enhance the adhesion between the sickle RBCs and macrophages. This, in turn, increases the retention of RBCs by as much as five-fold, which could be a possible cause of RBC congestion in the spleen of patients with SCD. Our study on the impact of RBC aggregation illustrates a 'clustering effect', where multiple RBCs in one aggregate can make contact and adhere to the macrophages, leading to a higher retention rate than that resulting from RBC-macrophage pair interactions. Our simulations of sickle RBCs flowing past macrophages for a range of blood flow velocities indicate that the increased blood velocity could quickly attenuate the function of the red pulp macrophages on detaining aged or diseased RBCs, thereby providing a possible rationale for the slow blood flow in the open circulation of the spleen. Furthermore, we quantify the impact of RBC morphology on their tendency to be retained by the macrophages. We find that the sickle and granular-shaped RBCs are more likely to be filtered by macrophages in the spleen. This finding is consistent with the observation of low percentages of these two forms of sickle RBCs in the blood smear of SCD patients. Taken together, our experimental and simulation results aid in our quantitative understanding of the function of splenic macrophages in retaining the diseased RBCs and provide an opportunity to combine such knowledge with the current knowledge of the interaction between IES and traversing RBCs to apprehend the complete filtration function of the spleen in SCD.

Rapid electrical impedance detection of sickle cell vaso-occlusion in microfluidic device.

Sickle cell disease is characterized by painful vaso-occlusive crises, in which poorly deformable sickle cells play an important role in the complex vascular obstruction process. Existing techniques are mainly based on optical microscopy and video processing of sickle blood flow under normoxic condition, for measuring vaso-occlusion by a small fraction of dense sickle cells of intrinsic rigidity but not the vaso-occlusion by the rigid, sickled cells due to deoxygenation. Thus, these techniques are not suitable for rapid, point-of-care testing. Here, we integrate electrical impedance sensing and Polydimethylsiloxane-microvascular mimics with controlled oxygen level into a single microfluidic chip, for quantification of vaso-occlusion by rigid, sickled cells within 1 min. Electrical impedance measurements provided a label-free, real-time detection of different sickle cell flow behaviors, including steady flow, vaso-occlusion, and flow recovery in response to the deoxygenation-reoxygenation process that are validated by microscopic videos. Sensitivity of the real part and imaginary part of the impedance signals to the blood flow conditions in both natural sickle cell blood and simulants at four electrical frequencies (10, 50, 100, and 500 kHz) are compared. The results show that the sensitivity of the sensor in detection of vaso-occlusion decreases as electrical frequency increases, while the higher frequencies are preferable in measurement of steady flow behavior. Additional testing using sickle cell simulants, chemically crosslinked normal red blood cells, shows same high sensitivity in detection of vaso-occlusion as sickle cell vaso-occlusion under deoxygenation. This work enables point-of-care testing potentials in rapid, accurate detection of steady flow and sickle cell vaso-occlusion from microliter volume blood specimens. Quantification of sickle cell rheology in response to hypoxia, may provide useful indications for not only the kinetics of cell sickling, but also the altered hemodynamics as obseved at the microcirculatory level.

In silico and in vitro study of the adhesion dynamics of erythrophagocytosis in sickle cell disease.

Erythrophagocytosis occurring in the spleen is a critical process for removing senescent and diseased red blood cells (RBCs) from the microcirculation. Although some progress has been made in understanding how the biological signaling pathways mediate the phagocytic processes, the role of the biophysical interaction between RBCs and macrophages, particularly under pathological conditions such as sickle cell disease, has not been adequately studied. Here, we combine computational simulations with microfluidic experiments to quantify RBC-macrophage adhesion dynamics under flow conditions comparable to those in the red pulp of the spleen. We also investigate the RBC-macrophage interaction under normoxic and hypoxic conditions. First, we calibrate key model parameters in the adhesion model using microfluidic experiments for normal and sickle RBCs under normoxia and hypoxia. We then study the adhesion dynamics between the RBC and the macrophage. Our simulation illustrates three typical adhesion states, each characterized by a distinct dynamic motion of the RBCs, namely firm adhesion, flipping adhesion, and no adhesion (either due to no contact with macrophages or detachment from the macrophages). We also track the number of bonds formed when RBCs and macrophages are in contact, as well as the contact area between the two interacting cells, providing mechanistic explanations for the three adhesion states observed in the simulations and microfluidic experiments. Furthermore, we quantify, for the first time to our knowledge, the adhesive forces between RBCs (normal and sickle) and macrophages under different oxygenated conditions. Our results show that the adhesive forces between normal cells and macrophages under normoxia are in the range of 33-58 pN and 53-92 pN for sickle cells under normoxia and 155-170 pN for sickle cells under hypoxia. Taken together, our microfluidic and simulation results improve our understanding of the biophysical interaction between RBCs and macrophages in sickle cell disease and provide a solid foundation for investigating the filtration function of the splenic macrophages under physiological and pathological conditions.

Microfluidic study of retention and elimination of abnormal red blood cells by human spleen with implications for sickle cell disease.

The spleen clears altered red blood cells (RBCs) from circulation, contributing to the balance between RBC formation (erythropoiesis) and removal. The splenic RBC retention and elimination occur predominantly in open circulation where RBCs flow through macrophages and inter-endothelial slits (IESs). The mechanisms underlying and interconnecting these processes significantly impact clinical outcomes. In sickle cell disease (SCD), blockage of intrasplenic sickled RBCs is observed in infants splenectomized due to acute splenic sequestration crisis (ASSC). This life-threatening RBC pooling and organ swelling event is plausibly triggered or enhanced by intra-tissular hypoxia. We present an oxygen-mediated spleen-on-a-chip platform for in vitro investigations of the homeostatic balance in the spleen. To demonstrate and validate the benefits of this general microfluidic platform, we focus on SCD and study the effects of hypoxia on splenic RBC retention and elimination. We observe that RBC retention by IESs and RBC-macrophage adhesion are faster in blood samples from SCD patients than those from healthy subjects. This difference is markedly exacerbated under hypoxia. Moreover, the sickled RBCs under hypoxia show distinctly different phagocytosis processes from those non-sickled RBCs under hypoxia or normoxia. We find that reoxygenation significantly alleviates RBC retention at IESs, and leads to rapid unsickling and fragmentation of the ingested sickled RBCs inside macrophages. These results provide unique mechanistic insights into how the spleen maintains its homeostatic balance between splenic RBC retention and elimination, and shed light on how disruptions in this balance could lead to anemia, splenomegaly, and ASSC in SCD and possible clinical manifestations in other hematologic diseases.

Computational modeling of biomechanics and biorheology of heated red blood cells.

Because of their compromised deformability, heat denatured erythrocytes have been used as labeled probes to visualize spleen tissue or to assess the ability of the spleen to retain stiff red blood cells (RBCs) for over three decades, e.g., see Looareesuwan et al. N. Engl. J. Med. (1987). Despite their good accessibility, it is still an open question how heated RBCs compare to certain diseased RBCs in terms of their biomechanical and biorheological responses, which may undermine their effective usage and even lead to misleading experimental observations. To help answering this question, we perform a systematic computational study of the hemorheological properties of heated RBCs with several physiologically relevant static and hemodynamic settings, including optical-tweezers test, relaxation of prestretched RBCs, RBC traversal through a capillary-like channel and a spleen-like slit, and a viscometric rheology test. We show that our in silico RBC models agree well with existing experiments. Moreover, under static tests, heated RBCs exhibit deformability deterioration comparable to certain disease-impaired RBCs such as those in malaria. For RBC traversal under confinement (through microchannel or slit), heated RBCs show prolonged transit time or retention depending on the level of confinement and heating procedure, suggesting that carefully heat-treated RBCs may be useful for studying splenic- or vaso-occlusion in vascular pathologies. For the rheology test, we expand the existing bulk viscosity data of heated RBCs to a wider range of shear rates (1-1000 s-1) to represent most pathophysiological conditions in macro- or microcirculation. Although heated RBC suspension shows elevated viscosity comparable to certain diseased RBC suspensions under relatively high shear rates (100-1000 s-1), they underestimate the elevated viscosity (e.g., in sickle cell anemia) at low shear rates (<10 s-1). Our work provides mechanistic rationale for selective usage of heated RBC as a potentially useful model for studying the abnormal traversal dynamics and hemorheology in certain blood disorders.

In vitro assay for single-cell characterization of impaired deformability in red blood cells under recurrent episodes of hypoxia.

Red blood cells (RBCs) are subjected to recurrent changes in shear stress and oxygen tension during blood circulation. The cyclic shear stress has been identified as an important factor that alone can weaken cell mechanical deformability. The effects of cyclic hypoxia on cellular biomechanics have yet to be fully investigated. As the oxygen affinity of hemoglobin plays a key role in the biological function and mechanical performance of RBCs, the repeated transitions of hemoglobin between its R (high oxygen tension) and T (low oxygen tension) states may impact their mechanical behavior. The present study focuses on developing a novel microfluidic-based assay for characterization of the effects of cyclic hypoxia on cell biomechanics. The capability of this assay is demonstrated by a longitudinal study of individual RBCs in health and sickle cell disease subjected to cyclic hypoxia conditions of various durations and levels of low oxygen tension. The viscoelastic properties of cell membranes are extracted from tensile stretching and relaxation processes of RBCs induced by the electrodeformation technique. Results demonstrate that cyclic hypoxia alone can significantly reduce cell deformability, similar to the fatigue damage accumulated through cyclic mechanical loading. RBCs affected by sickle cell disease are less deformable (significantly higher membrane shear modulus and viscosity) than normal RBCs. The fatigue resistance of sickle RBCs to the cyclic hypoxia challenge is significantly inferior to that of normal RBCs, and this trend is more significant in mature erythrocytes of sickle cells. When the oxygen affinity of sickle hemoglobin is enhanced by anti-sickling drug treatment of 5-hydroxymethyl-2-furfural (5-HMF), sickle RBCs show ameliorated resistance to fatigue damage induced by cyclic hypoxia. These results indicate an important biophysical mechanism underlying RBC senescence in which the cyclic hypoxia challenge alone can lead to mechanical degradation of the RBC membrane. We envision that the application of this assay can be further extended to RBCs in other blood diseases and other cell types.

A portable impedance microflow cytometer for measuring cellular response to hypoxia.

This article presents the development and testing of a low-cost (<$60), portable, electrical impedance-based microflow cytometer for single-cell analysis under a controlled oxygen microenvironment. The system is based on an AD5933 impedance analyzer chip, a microfluidic chip, and an Arduino microcontroller operated by a custom Android application. A representative case study on human red blood cells (RBCs) affected by sickle cell disease is conducted to demonstrate the capability of the cytometry system. Impedance values of sickle blood samples exhibit remarkable deviations from the common reference line obtained from two normal blood samples. Such deviation is quantified by a conformity score, which allows for the measurement of intrapatient and interpatient variations of sickle cell disease. A low conformity score under oxygenated conditions or drastically different conformity scores between oxygenated and deoxygenated conditions can be used to differentiate a sickle blood sample from normal. Furthermore, an equivalent circuit model of a suspended biological cell is used to interpret the electrical impedance of single flowing RBCs. In response to hypoxia treatment, all samples, regardless of disease state, exhibit significant changes in at least one single-cell electrical property, that is, cytoplasmic resistance and membrane capacitance. The overall response to hypoxia is less in normal cells than those affected by sickle cell disease, where the change in membrane capacitance varies from -23% to seven times as compared with -17% in normal cells. The results reported in this article suggest that the developed method of testing demonstrates the potential application for a low-cost screening technique for sickle cell disease and other diseases in the field and low-resource settings. The developed system and methodology can be extended to analyze cellular response to hypoxia in other cell types.

Dielectric spectroscopy of red blood cells in sickle cell disease.

Hypoxia-induced polymerization of sickle hemoglobin and the related ion diffusion across cell membrane can lead to changes in cell dielectric properties, which can potentially serve as label-free, diagnostic biomarkers for sickle cell disease. This article presents a microfluidic-based approach with on-chip gas control for the impedance spectroscopy of suspended cells within the frequency range of 40 Hz to 110 MHz. A comprehensive bioimpedance of sickle cells under both normoxia and hypoxia is achieved rapidly (within ∼7 min) and is appropriated by small sample volumes (∼2.5 µL). Analysis of the sensing modeling is performed to obtain optimum conditions for dielectric spectroscopy of sickle cell suspensions and for extraction of single cell properties from the measured impedance spectra. The results of sickle cells show that upon hypoxia treatment, cell interior permittivity and conductivity increase, while cell membrane capacitance decreases. Moreover, the relative changes in cell dielectric parameters are found to be dependent on the sickle and fetal hemoglobin levels. In contrast, the changes in normal red blood cells between the hypoxia and normoxia states are unnoticeable. The results of sickle cells may serve as a reference to design dielectrophoresis-based cell sorting and electrodeformation testing devices that require cell dielectric characteristics as input parameters. The demonstrated method for dielectric characterization of single cells from the impedance spectroscopy of cell suspensions can be potentially applied to other cell types and under varied gas conditions.

Rapid Characterization of Water Diffusion in Polymer Specimens Using a Droplet-Based Method.

Water diffusion testing is typically carried out by immersing specimens in a water bath and monitoring water uptake until saturation is reached. Determination of diffusivity may require several months and even years for thick specimens. In this paper, we present a water droplet-based method for rapid characterization of diffusivity. The method involves placement of a water droplet on a flat surface of the testing material. A tensiometer is used to monitor and record the evaluation of droplet dimensions. The small volume of the water droplet (below 10 µL) ensures that diffusivity can be determined in a couple of hours. The capability of this method is demonstrated by determining the water diffusion (D) of polymethylmethacrylate (PMMA) and epoxy plastics. The water diffusivity measured for PMMA matched well with published results. The droplet method was also applied to void-free epoxy and epoxy with a range of void contents. The diffusivity for the epoxy with voids increased with increasing void content. The diffusivity results for the epoxy without voids and with small void content agree with those determined from the long-term water immersion method. For the high-void-content epoxy, the diffusivity was much higher than that in the immersion method. This may be because of the rough surface caused by large exposed voids.

Optimization of in vitro trophoblast assay for real-time impedimetric sensing of trophoblast-erythrocyte interactions in Plasmodium falciparum malaria.

Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) is responsible for the pathophysiology of placental malaria, leading to serious complications such as intrauterine growth restriction and low birth weight. However, it is an experimental challenge to study the biology of human placenta. Conventional cell culture-based in vitro placental models rely on immunostaining techniques and high-magnification microscopy is limited in providing real-time quantitative analysis. Impedimetric sensing in combination with cell culture may offer a useful tool. In this paper, we report that real-time label-free measurement of cellular electrical impedance using xCELLigence technology can be used to quantify the proliferation, syncytial fusion, and long-term response of BeWo cells to IEs cytoadhesion. Specifically, we optimized key experimental parameters of cell seeding density and concentration of forskolin, a compound used to promote cell syncitiation, based on electrical signals and immunostaining results. Prolonged time of infection with IEs that led to cell-cell junction vanishment in BeWo cells and release of inflammatory cytokines were monitored in real time by continuous change in electrical impedance. The results suggest that the impedimetric technique is sensitive and can offer new opportunities for the study of cellular responses of trophoblast cells to IEs. The developed system can provide potentially a high-throughput screening tool of anti-adhesion or anti-inflammatory drugs for placental malaria infections.

Mechanical fatigue of human red blood cells.

Fatigue arising from cyclic straining is a key factor in the degradation of properties of engineered materials and structures. Fatigue can also induce damage and fracture in natural biomaterials, such as bone, and in synthetic biomaterials used in implant devices. However, the mechanisms by which mechanical fatigue leads to deterioration of physical properties and contributes to the onset and progression of pathological states in biological cells have hitherto not been systematically explored. Here we present a general method that employs amplitude-modulated electrodeformation and microfluidics for characterizing mechanical fatigue in single biological cells. This method is capable of subjecting cells to static loads for prolonged periods of time or to large numbers of controlled mechanical fatigue cycles. We apply the method to measure the systematic changes in morphological and biomechanical characteristics of healthy human red blood cells (RBCs) and their membrane mechanical properties. Under constant amplitude cyclic tensile deformation, RBCs progressively lose their ability to stretch with increasing fatigue cycles. Our results further indicate that loss of deformability of RBCs during cyclic deformation is much faster than that under static deformation at the same maximum load over the same accumulated loading time. Such fatigue-induced deformability loss is more pronounced at higher amplitudes of cyclic deformation. These results uniquely establish the important role of mechanical fatigue in influencing physical properties of biological cells. They further provide insights into the accumulated membrane damage during blood circulation, paving the way for further investigations of the eventual failure of RBCs causing hemolysis in various hemolytic pathologies.

Electrical Impedance Characterization of Erythrocyte Response to Cyclic Hypoxia in Sickle Cell Disease.

Cell sickling is the process in which intracellular polymerization of deoxygenated sickle hemoglobin (HbS) leads to distorted, rigid cells, resulting in abnormal blood rheology and painful vaso-occlusion. Current methods for detection of this process mainly rely on optical microscopy of cellular morphology and measurements of cell deformability and blood rheology. As electrical impedance of cells is a sensitive indicator of changes in cellular structure and biophysical characteristics, it can be a promising marker for characterization of abnormal blood rheology and a means more convenient than optics to be integrated into point-of-care devices. In this work, a microfluidics-based electrical impedance sensor has been developed for characterizing the dynamic cell sickling-unsickling processes in sickle blood. The sensor is capable of measuring the continuous variation in the sickle cell suspension due to cyclic hypoxia-induced intracellular HbS polymerization and depolymerization. Simultaneous microscopic imaging of cell morphological change shows the reliability and repeatability of the electrical impedance-based measurements of cell sickling and unsickling processes. Strong correlation is found between the electrical impedance measurement and patients' hematological parameters such as levels of HbS and fetal hemoglobin. The combination of electrical impedance measurement and on-chip hypoxia control provides a promising method for rapid assessment of the dynamic processes of cell sickling and unsickling in patients with sickle cell disease.

Modeling erythrocyte electrodeformation in response to amplitude modulated electric waveforms.

We present a comprehensive theoretical-experimental framework for quantitative, high-throughput study of cell biomechanics. An improved electrodeformation method has been developed by combing dielectrophoresis and amplitude shift keying, a form of amplitude modulation. This method offers a potential to fully control the magnitude and rate of deformation in cell membranes. In healthy human red blood cells, nonlinear viscoelasticity of cell membranes is obtained through variable amplitude load testing. A mathematical model to predict cellular deformations is validated using the experimental results of healthy human red blood cells subjected to various types of loading. These results demonstrate new capabilities of the electrodeformation technique and the validated mathematical model to explore the effects of different loading configurations on the cellular mechanical behavior. This gives it more advantages over existing methods and can be further developed to study the effects of strain rate and loading waveform on the mechanical properties of biological cells in health and disease.

Electrical impedance microflow cytometry with oxygen control for detection of sickle cells.

Polymerization of intracellular sickle hemoglobin induced by low oxygen tension has been recognized as a primary determinant of the pathophysiologic manifestations in sickle cell disease. Existing flow cytometry techniques for detection of sickle cells are typically based on fluorescence markers or cellular morphological analysis. Using microfluidics and electrical impedance spectroscopy, we develop a new, label-free flow cytometry for non-invasive measurement of single cells under controlled oxygen level. We demonstrate the capability of this new technique by determining the electrical impedance differential of normal red blood cells obtained from a healthy donor and sickle cells obtained from three sickle cell patients, under normoxic and hypoxic conditions and at three different electrical frequencies, 156 kHz, 500 kHz and 3 MHz. Under normoxia, normal cells and sickle cells can be separated completely using electrical impedance at 156 kHz and 500 kHz but not at 3 MHz. Sickle cells, intra-patient and inter-patient show significantly different electrical impedance between normoxia and hypoxia at all three frequencies. This study shows a proof of concept that electrical impedance signal can be used as an indicator of the disease state of a red blood cell as well as the cell sickling events in sickle cell disease. Electrical impedance-based microflow cytometry with oxygen control is a new method that can be potentially used for sickle cell disease diagnosis and monitoring.

Dielectrophoresis Testing of Nonlinear Viscoelastic Behaviors of Human Red Blood Cells.

Dielectrophoresis in microfluidics provides a useful tool to test biomechanics of living cells, regardless of surface charges on cell membranes. We have designed an experimental method to characterize the nonlinear viscoelastic behaviors of single cells using dielectrophoresis in a microfluidic channel. This method uses radio frequency, low voltage excitations through interdigitated microelectrodes, allowing probing multiple cells simultaneously with controllable load levels. Dielectrophoretic force was calibrated using a triaxial ellipsoid model. Using a Kelvin-Voigt model, the nonlinear shear moduli of cell membranes were determined from the steady-state deformations of red blood cells in response to a series of electric field strengths. The nonlinear elastic moduli of cell membranes ranged from 6.05 µN/m to up to 20.85 µN/m, which were identified as a function of extension ratio, rather than the lumped-parameter models as reported in the literature. Value of the characteristic time of the extensional recovery of cell membranes initially deformed to varied extent was found to be about 0.14 s. Shear viscosity of cell membrane was estimated to be 0.8-2.9 (µN/m)·s. This method is particularly valuable for rapid, non-invasive probing of mechanical properties of living cells.

Erythrocyte Membrane Failure by Electromechanical Stress.

We envision that electrodeformation of biological cells through dielectrophoresis as a new technique to elucidate the mechanistic details underlying membrane failure by electrical and mechanical stresses. Here we demonstrate the full control of cellular uniaxial deformation and tensile recovery in biological cells via amplitude-modified electric field at radio frequency by an interdigitated electrode array in microfluidics. Transient creep and cyclic experiments were performed on individually tracked human erythrocytes. Observations of the viscoelastic-to-viscoplastic deformation behavior and the localized plastic deformations in erythrocyte membranes suggest that electromechanical stress results in irreversible membrane failure. Examples of membrane failure can be separated into different groups according to the loading scenarios: mechanical stiffening, physical damage, morphological transformation from discocyte to echinocyte, and whole cell lysis. These results show that this technique can be potentially utilized to explore membrane failure in erythrocytes affected by other pathophysiological processes.

Dynamic fatigue measurement of human erythrocytes using dielectrophoresis.

Erythrocytes must undergo severe deformation to pass through narrow capillaries and submicronic splenic slits for several hundred thousand times in their normal lifespan. Studies of erythrocyte biomechanics have been mainly focused on cell deformability and rheology measured from a single application of stress and mostly under a static or quasi-static state using classical biomechanical techniques, such as optical tweezers and micropipette aspiration. Dynamic behavior of erythrocytes in response to cyclic stresses that contributes to the membrane failure in blood circulation is not fully understood. This paper presents a new experimental method for dynamic fatigue analysis of erythrocytes, using amplitude modulated electrokinetic force field in a microfluidic platform. We demonstrate the capability of this new technique using a low cycle fatigue analysis of normal human erythrocytes and ATP-depleted erythrocytes. Cyclic tensile stresses are generated to induce repeated uniaxial stretching and extensional recovery of single erythrocytes. Results of morphological and biomechanical parameters of individually tracked erythrocytes show strong correlations with the number of the loading cycles. Under a same strength of electric field, after 180 stress cycles, for normal erythrocytes, maximum stretch ratio decreases from 3.80 to 2.86, characteristic time of cellular extensional recovery increases from 0.16s to 0.37s, membrane shear viscosity increases from 1.0(µN/m)s to 1.6(µN/m)s. Membrane deformation in a small number of erythrocytes becomes irreversible after large deformation for about 200 cyclic loads. ATP-depleted cells show similar trends in decreased deformation and increased characteristic time with the loading cycles. These results show proof of concept of the new microfluidics technique for dynamic fatigue analysis of human erythrocytes. STATEMENT OF SIGNIFICANCE: Red blood cells (RBCs) experience a tremendous number of deformation in blood circulation before losing their mechanical deformability and eventually being degraded in the reticuloendothelial system. Prior efforts in RBC biomechanics have mostly focused on a single-application of stress, or quasi-static loading through physical contact to deform cell membranes, thus with limited capabilities in probing cellular dynamic responses to cyclic stresses. We present a unique electrokinetic microfluidic system for the study of dynamic fatigue behavior of RBCs subjected to cyclic loads. Our work shows quantitatively how the cyclic stretching loads cause membrane mechanical degradation and irreversibly deformed cells. This new technique can be useful to identify biomechanical markers for prediction of the mechanical stability and residual lifespan of circulating RBCs.