Radial displacement of red blood cells during hemodilution and the effect on arteriolar oxygen profile

In this study, we assessed the magnitude of the erratic deviations in the radial position of red blood cells (RBCs) in the laminar flow regime of arterioles in a hamster window preparation and the intraluminal Po(2) profile to determine whether this variability affects the intraluminal distribution of oxygen in conditions of normal hematocrit and hemodilution. A gated image intensifier was used to visualize fluorescently labeled RBCs in tracer quantities and obtain multiple measurements of RBC radial and longitudinal positions at time intervals on the order of 5 ms within single arterioles (diameter range 40-95 microm). RBCs in the velocity range of 0.3-14 mm/s exhibit a mean coefficient of variation of velocity of 16.9 +/- 10.5% and a SD of the radial position of 1.98 +/- 0.98 microm. Both quantities were inversely related to hematocrit, and the former was significantly lowered by hemodilution. Our experimental results presented very similar values and shape compared with the intraluminal oxygen profile derived theoretically for normal hematocrit, suggesting that shear-augmented diffusion due to the measured radial displacement of RBCs did not significantly affect oxygen diffusion from blood into the arteriolar vessel wall. Po(2) profiles in the arterioles assumed an increasingly parabolic configuration with increasing levels of hemodilution.     

In vivo full‐field functional optical hemocytometer

We present an in vivo lab‐free full‐field functional optical hemocytometer (FFOH) for application to the capillaries of a live biological specimen, based on the absorption intensity fluctuation modulation (AIFM) effect. Because of the absorption difference between the red blood cells (RBCs) and background tissue under low‐coherence light illumination, an endogenous instantaneous intensity fluctuation is generated by the AIFM effect when RBCs discontinuously traverse the capillary. The AIFM effect is used to highlight the RBC signal relative to the background tissue by computing the real‐time modulation depth. FFOH can simultaneously provide a flow video, the flow velocity and the RBC count. Ourexperimental results can potentially be applied to study the physiological mechanisms of the blood circulation systems of near‐transparent live biological samples.      

Optofluidic single‐cell absorption flow analyzer for point‐of‐care diagnosis of malaria

In this work, an optofluidic flow analyzer, which can be used to perform malaria diagnosis at the point‐of‐care is demonstrated. The presented technique is based on quantitative optical absorption measurements carried out on a single cell level for a given population of Human Red Blood Cells (RBCs). By measuring the optical absorption of each RBC, the decrease in the Hemoglobin (Hb) concentration in the cytoplasm of the cell due to the invasion of malarial parasite is detected. Cells are assessed on a single cell basis, as they pass through a microfluidic channel. The proposed technique has been implemented with inexpensive off‐the‐shelf components like laser diode, photo‐detector and a micro‐controller. The ability of the optofluidic flow analyzer to asses about 308,049 cells within 3 minutes has been demonstrated. The presented technique is capable of detecting very low parasitemia levels with high sensitivity.

     

In vivo micro-circulation measurement in skeletal muscle by intra-vital microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman''s method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized, ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT & CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.Download video file.^{(92M, mpg)}     

Electrochemical analysis of blood cell/substrate interactions under flow conditions

Interactions of blood cells (RBCs) with a microelectrode of 50 (im diameter have been examined under flow conditions using impedance measurements at high frequencies. At such frequencies, the electrolyte resistance (R_e,) is assimilated to the real part of impedance, and interactions are associated with transient fluctuations of R_e. Sedimentation experiments suggest that one erythrocyte contributes to a 1.1% R_e, increase. Effects of wall shear rate (from 25 to 140 s¹) and RBC concentration (from 8.4 × 10⁵ to 2.7 × 10⁶ cells/ml) have been investigated; the number of interactions rapidly decreases with wall shear rate. Event frequency is proportional to RBC concentration ranging from 3.1 × 10⁶ cells/ml to 1.3 × 10⁷ cells/ml. At high concentrations of RBCs, some transient events overlap. Videotaped images help to determine how many RBCs interact with the microelectrode at the same time on separate surface areas. Under flow conditions, the contribution of one RBC on the R_e increase is similar to the mathematical value obtained by sedimentation and decreases slightly with wall shear rate.     

Resting‐state functional connectivity measured by diffuse correlation spectroscopy

Near‐infrared diffuse correlation spectroscopy (DCS) is used to record spontaneous cerebral blood flow fluctuations in the frontal cortex. Nine adult subjects participated in the experiments, in which 8‐minute spontaneous fluctuations were simultaneously recorded from the left and right dorsolateral and inferior frontal regions. Resting‐state functional connectivity (RSFC) was measured by the temporal correlation of the low frequency fluctuations. Our data shows the RSFC within the dorsolateral region is significantly stronger than that between the inferior and dorsolateral regions, in line with previous observations with functional near‐infrared spectroscopy. This indicates that DCS is capable of investigating brain functional connectivity in terms of cerebral blood flow.      

An automated cell analysis sensing system based on a microfabricated rheoscope for the study of red blood cells physiology

An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. RBCs suspended in a high viscosity medium were filmed flowing through a microchannel. Under these conditions, RBCs exhibit different orientations and deformations according to their location in the velocity profile. The rheoscope system produces valuable data such as velocity profile of RBCs, spatial distribution within a microchannel and deformation index (DI) curves. The variation of DI across the channel height, due to change in shear stress, was measured carrying implications for diffractometry methods. These curves of DI were taken at a constant flow rate and cover most of the relevant shear stress spectrum. This is an improvement of the existing techniques for deformability measurements and may serve as a diagnostic tool for certain blood disorders. The DI curves were compared to measurements of the flowing RBCs velocity profile. In addition, we found that RBCs flowing in a microchannel are mostly gathered in the center of the flow and maintain a characteristic spatial distribution. The spatial distribution in this region changes slightly with increasing flow rate. Hence, the system described, provides means for examining the behavior of individual RBCs, and may serve as a microfabricated diagnostic device for deformability measurement.     

One‐to‐one registration of en‐face optical coherence tomography attenuation coefficients with histology of a prostatectomy specimen

Optical coherence tomography (OCT), enables high‐resolution 3D imaging of the morphology of light scattering tissues. From the OCT signal, parameters can be extracted and related to tissue structures. One of the quantitative parameters is the attenuation coefficient; the rate at which the intensity of detected light decays in depth. To couple the quantitative parameters with the histology one‐to‐one registration is needed. The primary aim of this study is to validate a registration method of quantitative OCT parameters to histological tissue outcome through one‐to‐one registration of OCT with histology. We matched OCT images of unstained fixated prostate tissue slices with corresponding histology slides, wherein different histologic types were demarcated. Attenuation coefficients were determined by a supervised automated exponential fit (corrected for point spread function and sensitivity roll‐off related signal losses) over a depth of 0.32 mm starting from 0.10 mm below the automatically detected tissue edge. Finally, the attenuation coefficients corresponding to the different tissue types of the prostate were compared. From the attenuation coefficients, we produced the squared relative residue and goodness‐of‐fit metric R². This article explains the method to perform supervised automated quantitative analysis of OCT data, and the one‐to‐one registration of OCT extracted quantitative data with histopathological outcomes.      

Analysis of a high‐throughput cone‐and‐plate apparatus for the application of defined spatiotemporal flow to cultured cells

The shear stresses derived from blood flow regulate many aspects of vascular and immunobiology. In vitro studies on the shear stress‐mediated mechanobiology of endothelial cells have been carried out using systems analogous to the cone‐and‐plate viscometer in which a rotating, low‐angle cone applies fluid shear stress to cells grown on an underlying, flat culture surface. We recently developed a device that could perform high‐throughput studies on shear‐mediated mechanobiology through the rotation of cone‐tipped shafts in a standard 96‐well culture plate. Here, we present a model of the three‐dimensional flow within the culture wells with a rotating, cone‐tipped shaft. Using this model we examined the effects of modifying the design parameters of the system to allow the device to create a variety of flow profiles. We first examined the case of steady‐state flow with the shaft rotating at constant angular velocity. By varying the angular velocity and distance of the cone from the underlying plate we were able to create flow profiles with controlled shear stress gradients in the radial direction within the plate. These findings indicate that both linear and non‐linear spatial distributions in shear stress can be created across the bottom of the culture plate. In the transition and “parallel shaft” regions of the system, the angular velocities needed to provide high levels of physiological shear stress (5 Pa) created intermediate Reynolds number Taylor‐Couette flow. In some cases, this led to the development of a flow regime in which stable helical vortices were created within the well. We also examined the system under oscillatory and pulsatile motion of the shaft and demonstrated minimal time lag between the rotation of the cone and the shear stress on the cell culture surface. Biotechnol. Bioeng. 2013; 110: 1782–1793. © 2013 Wiley Periodicals, Inc.     

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.     

A photometric method to analyze induced erythrocyte shape changes

In this paper, a photometric method was introduced to quantify biochemically-induced red blood cell (RBC) shape changes when no shear force was acting on the cells. To obtain the photometric RBC shape parameter (RF1), a monolayer of point-attached RBCs was prepared on the floor of a flat flow chamber and the transmission of light perpendicular to the monolayer plane was measured: 1) in phosphate buffered saline with 0.1% bovine serum albumin (PBS₊) and 2) in PBS₊, containing a shape changing compound (in both, the RBCs were not deformed due to shear flow). To normalize the data, a third transmission value at a shear stress of 3 Pa was measured in PBS₊ from the same RBC monolayer. To validate the photometric data, RF1 of RBCs exposed to shape changing agents was correlated by linear regression analysis with 1) data obtained with the tangent-counting technique (TC) and 2) the morphological index (MI). The coefficient of correlation was calculated at 0.95 for the TC data and 0.94 for the MI data, respectively. The sensitivity of the photometric method was tested with stomatocytogenic chlorpromazine (CP) and echinocytogenic sodium salicylate (SA). CP (2.5 μM) induced a significant decrease of RF1 to −0.045 (N = 6 donors, p < 0.01), whereas SA (2.5 mM) increased RF1 to +0.027 significantly (N = 6, donors, p < 0.01). Both the CP-induced and the SA-induced shape changes appeared less than 2 min after application of the shape changing agents, and changed gradually within another 30 min when the agent was present in PBS₊, partly disappearing within about 2 min after reincubation of the shape transformed RBCs in PBS₊ not containing the agent.     

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.     

Effect of aggregation and shear rate on the dispersion of red blood cells flowing in venules

Previous in vitro studies of blood flow in small glass tubes have shown that red blood cells exhibit significant erratic deviations in the radial position in the laminar flow regime. The purpose of the present study was to assess the magnitude of this variability and that of velocity in vivo and the effect of red blood cell aggregation and shear rate upon them. With the use of a gated image intensifier and fluorescently labeled red blood cells in tracer quantities, we obtained multiple measurements of red blood cell radial and longitudinal positions at time intervals as short as 5 ms within single venous microvessels (diameter range 45-75 microm) of the rat spinotrapezius muscle. For nonaggregating red blood cells in the velocity range of 0.3-14 mm/s, the mean coefficient of variation of velocity was 16.9 +/- 10.5% and the SD of the radial position was 1.98 +/- 0.98 microm. Both quantities were inversely related to shear rate, and the former was significantly lowered on induction of red blood cell aggregation by the addition of Dextran 500 to the blood. The shear-induced random movements observed in this study may increase the radial transport of particles and solutes within the bloodstream by orders of magnitude.     

In vivo detection of endotracheal tube biofilms in intubated critical care patients using catheter‐based optical coherence tomography

The formation of biofilms in the endotracheal tubes (ETTs) of intubated patients on mechanical ventilation is associated with a greater risk of ventilator‐associated pneumonia and death. New technologies are needed to detect and monitor ETTs in vivo for the presence of these biofilms. Longitudinal OCT imaging was performed in mechanically ventilated subjects at 24‐hour intervals until extubation to detect the formation and temporal changes of in vivo ETT biofilms. OCT‐derived attenuation coefficient images were used to differentiate between mucus and biofilm. Extubated ETTs were examined with optical and electron microscopy, and all imaging results were correlated with standard‐of‐care clinical test reports. OCT and attenuation coefficient images from four subjects were positive for ETT biofilms and were negative for two subjects. The processed and stained extubated ETTs and clinical reports confirmed the presence/absence of biofilms in all subjects. Our findings confirm that OCT can detect and differentiate between biofilm‐positive and biofilm‐negative groups (P < 10^?5). OCT image‐based features may serve as biomarkers for direct in vivo detection of ETT biofilms and help drive investigation of new management strategies to reduce the incidence of VAP.      

Heart‐beat‐phase‐coherent Doppler optical coherence tomography for measuring pulsatile ocular blood flow

We introduce a Doppler OCT (DOCT) platform that is fully synchronized with the heart‐beat via a pulse oximeter. The system allows reconstructing heart‐beat‐phase‐coherent quantitative DOCT volumes. The method is to acquire a series of DOCT volumes and to record the pulse in parallel. The heartbeat data is used for triggering the start of each DOCT volume acquisition. The recorded volume series is registered to the level of capillaries using a cross‐volume registration. The information of the pulse phase is used to rearrange the tomograms in time, to obtain a series of phase coherent DOCT volumes over a pulse. We present Doppler angle independent quantitative evaluation of the absolute pulsatile blood flow within individual retinal vessels as well as of the total retinal blood flow over a full heartbeat cycle. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

On transport of suspended particulates in tube flow.

Blood cells suspended in shear flows exhibit much larger dispersive motions than those predicted by the Stokes-Einstein formula for Brownian diffusion. The lateral migration and the erratic motions of the 8 microns red blood cells (RBC) is thought to be analogous to a diffusive process. It is shown that the often cited convective-diffusion theory may not be an adequate model for describing the transverse migration of suspended cells in blood flow. A comprehensive review of both the classical theory and of contemporary work in particle transport is presented, with particular emphasis on low Reynolds number tube flows. The mechanisms of Taylor dispersion, the effects of Brownian perturbations on translational and rotational motions of the suspended particles in shear fields, and the influence of integratable and chaotic advections, are individually examined. The classical experiment by Segre and Silberberg (1962) lead us to believe that particle hydrodynamics may play an important role in transverse migrations. In this light, we have further examined the hydrodynamic aspects of the so-called "tubular pinch" effect, the lateral migration of rigid spheres. We have also discussed the transverse motions of liquid drops, and the reversibility of the organization of suspensions in transport. The convective accelerations in the entrance region of a tube can produce relative velocities between fluid medium and various type of particulates if there is a difference in density. The deformable RBC, an "active-type" particle, can provide feedback to the flow from both mass and momentum considerations; the more rigid platelet, a "passive-type" particle, will experience a much smaller relative velocity as compared to the RBC. We may expect that particles of different densities are transported to different equilibrium annular positions before entering the fully developed flow region. The erratic, lateral movement of suspended particulates in steady laminar tube flow can be described by the usual Lagrangian coordinates.     

Extracellular methemoglobin promotes cyto-adherence of uninfected RBC to endothelial cells: Insight into cerebral malaria pathology

The endothelial cell barrier is tightly regulated, and disruption or the leaky behavior of the barrier leads to pathology. Disturbance of blood-brain barrier is observed during viral infection, cerebral malaria, and acute hemorrhagic encephalitis. Red blood cells (RBCs) bind to the endothelial cells (ECs) and their affinity towards ECs enhances in the presence of Plasmodium falciparum infection. ECs stimulated with methemoglobin (MetHb; 20 µM) for 1 hour exhibit high levels of cyto-adherence receptors CD36 and ICAM-1 on their cell surface compared with unstimulated cells. These ECs have acquired affinity towards uninfected RBCs in flow at arterial shear stress. SEM analysis indicates that EC–RBC cyto-adherence involved multiple attachment points. Initially, ECs bind single layer of RBCs and the number of RBCs increases over time to give high-order cyto-adherence with more than 30 RBCs adhered to each endothelial cell. The cyto-adherence complexes are stable to high shear stress and can withstand shear stress up to 450 dyne/cm ². MetHb-treated ECs exhibited high reactive oxygen species level, and preincubation of ECs with antioxidant (NAC or mannitol) abolished the formation of EC–RBC cyto-adherence complexes. In addition, gallic acid (present in red wine) and green tea extract has inhibited the formation of EC–RBC cyto-adherence complex. A better understanding of gallic acid and tea polyphenol targeting pathological cyto-adherence may allow us to develop a better adjuvant therapy for cerebral malaria and other noninfectious diseases.     

Laminar shear stress elicit distinct endothelial cell e‐selectin expression pattern via TNFα and IL‐1β activation

The ability to discriminate cell adhesion molecule expression between healthy and inflamed endothelium is critical for therapeutic intervention in many diseases. This study explores the effect of laminar flow on TNFα‐induced E‐selectin surface expression levels in human umbilical vein endothelial cells (HUVECs) relative to IL‐1β‐induced expression via flow chamber assays. HUVECs grown in static culture were either directly (naïve) activated with cytokine in the presence of laminar shear or pre‐exposed to 12 h of laminar shear (shear‐conditioned) prior to simultaneous shear and cytokine activation. Naïve cells activated with cytokine in static served as control. Depending on the cell shear history, fluid shear is found to differently affect TNFα‐induced relative to IL‐1β‐induced HUVEC expression of E‐selectin. Specifically, E‐selectin surface expression by naïve HUVECs is enhanced in the 8–12 h activation time range with simultaneous exposure to shear and TNFα (shear‐TNFα) relative to TNFα static control whereas enhanced E‐selectin expression is observed in the 4–24 h range for shear‐IL‐1β treatment relative to IL‐1β static control. While exposure of HUVECs to shear preconditioning mutes shear‐TNFα‐induced E‐selectin expression, it enhances or down‐regulates shear‐IL‐1β‐induced expression dependent on the activation period. Under dual‐cytokine‐shear conditions, IL‐1β signaling dominates. Overall, a better understanding of E‐selectin expression pattern by human ECs relative to the combined interaction of cytokines, shear profile and history can help elucidate many disease pathologies. Biotechnol. Bioeng. 2013; 110: 999–1003. © 2012 Wiley Periodicals, Inc.