Partitioning of dense RBC suspensions in single microfluidic bifurcations: role of cell deformability and bifurcation angle.

Red blood cells (RBCs) are a key determinant of human physiology and their behaviour becomes extremely heterogeneous as they navigate in narrow, bifurcating vessels in the microvasculature, affecting local haemodynamics. This is due to partitioning in bifurcations which is dependent on the biomechanical properties of RBCs, especially deformability. We examine the effect of deformability on the haematocrit distributions of dense RBC suspensions flowing in a single, asymmetric Y-shaped bifurcation, experimentally. Human RBC suspensions (healthy and artificially hardened) at 20% haematocrit (Ht) were perfused through the microchannels at different flow ratios between the outlet branches, and negligible inertia, and imaged to infer cell distributions. Notable differences in the shape of the haematocrit distributions were observed between healthy and hardened RBCs near the bifurcation apex. These lead to more asymmetric distributions for healthy RBCs in the daughter and outlet branches with cells accumulating near the inner channel walls, exhibiting distinct hematocrit peaks which are sharper for healthy RBCs. Although the hematocrit distributions differed locally, similar partitioning characteristics were observed for both suspensions. Comparisons with RBC distributions measured in a T-shaped bifurcation showed that the bifurcation angle affects the haematocrit characteristics of the healthy RBCs and not the hardened ones. The extent of RBC partitioning was found similar in both geometries and suspensions. The study highlights the differences between local and global characteristics which impact RBC distribution in more complex, multi-bifurcation networks.

Red blood cell sedimentation rate measurements in a high aspect ratio microchannel.

BACKGROUND: The erythrocyte sedimentation rate (ESR) test is commonly used in clinical practice for monitoring, screening and diagnosing pathological conditions and diseases related to the inflammatory response of the immune system. Several ESR techniques have been developed over the years improving the reliability, the precision and the duration of the measurement. OBJECTIVE: In the present study a new low cost micro-ESR technique is described providing the major advantage of reducing the measurement time and the blood sample volume by multiple times compared to the commercial methods. METHODS: Blood samples were obtained from healthy donors within the age group of 24-28 years and the haematocrit was adjusted to 30%, 40% and 50%. The ESR of the samples was measured utilizing a surface tension driven (STD) microfluidic chip and a monitoring device. RESULTS: The evaluation of the method showed a high correlation (0.94, p < 10-5) at all haematocrit levels with the commercial instrument indicating the feasibility of the technique. CONCLUSIONS: This micro-ESR technique provides the potential for a simple, low cost and fast tool for ESR measurement using low blood volume acquired by finger prick.

Enhanced biodegradation and valorization of drilling wastewater via simultaneous production of biosurfactants and polyhydroxyalkanoates by Pseudomonas citronellolis SJTE-3.

Pseudomonas citronellolis SJTE-3 was isolated as a highly efficient microorganism for biodegradation and valorization of drilling fluids (DF) wastewater. The strain metabolised DF and oily mud exhibiting up to 93%, 86%, 85% and 88% of chemical oxygen demand (COD), n-dodecane, n-tetradecane and naphthalene removal efficiency respectively. Enhanced bioconversion was enabled through production of biosurfactants that reduced the surface tension of water by 53% and resulted in 43.3% emulsification index (E24), while synthesizing 24% of dry cell weight (DCW) as medium-chain-length polyhydroxyalkanoates (PHA). Expression from the main pathways for alkanes and naphthalene biodegradation as well as biosurfactants and PHA biosynthesis revealed that although the alkanes and naphthalene biodegradation routes were actively expressed even at stationary phase, PHA production was stimulated at late stationary phase and putisolvin could comprise the biosurfactant synthesized. The bioconversion of toxic petrochemical residues to added-value thermoelastomers and biosurfactants indicate the high industrial significance of P. citronellolis SJTE-3.

Development of an Optical Method for the Evaluation of Whole Blood Coagulation.

Blood coagulation is a defense mechanism, which is activated in case of blood loss, due to vessel damage, or other injury. Pathological cases arise from malfunctions of the blood coagulation mechanism, and rapid growth of clots results in partially or even fully blocked blood vessel. The aim of this work is to characterize blood coagulation, by analyzing the time-dependent structural properties of whole blood, using an inexpensive design and robust processing approaches. The methods used in this work include brightfield microscopy and image processing techniques, applied on finger-prick blood samples. The blood samples were produced and directly utilized in custom-made glass microchannels. Color images were captured via a microscopy-camera setup for a period of 35 min, utilizing three different magnifications. Statistical information was extracted directly from the color components and the binary conversions of the images. The main advantage in the current work lies on a Boolean classification approach utilized on the binary data, which enabled to identify the interchange between specific structural elements of blood, namely the red blood cells, the plasma and the clotted regions, as a result of the clotting process. Coagulation indices produced included a bulk coagulation index, a plasma-reduction based index and a clot formation index. The results produced with the inexpensive design and the low computational complexity in the current approach, show good agreement with the literature, and a great potential for a robust characterization of blood coagulation.

An Investigation on the Aggregation and Rheodynamics of Human Red Blood Cells Using High Performance Computations.

Studies on the haemodynamics of human circulation are clinically and scientifically important. In order to investigate the effect of deformation and aggregation of red blood cells (RBCs) in blood flow, a computational technique has been developed by coupling the interaction between the fluid and the deformable RBCs. Parallelization was carried out for the coupled code and a high speedup was achieved based on a spatial decomposition. In order to verify the code's capability of simulating RBC deformation and transport, simulations were carried out for a spherical capsule in a microchannel and multiple RBC transport in a Poiseuille flow. RBC transport in a confined tube was also carried out to simulate the peristaltic effects of microvessels. Relatively large-scale simulations were carried out of the motion of 49,512 RBCs in shear flows, which yielded a hematocrit of 45%. The large-scale feature of the simulation has enabled a macroscale verification and investigation of the overall characteristics of RBC aggregations to be carried out. The results are in excellent agreement with experimental studies and, more specifically, both the experimental and simulation results show uniform RBC distributions under high shear rates (60-100/s) whereas large aggregations were observed under a lower shear rate of 10/s.

Quantifying local characteristics of velocity, aggregation and hematocrit of human erythrocytes in a microchannel flow.

The effect of erythrocyte aggregation on blood viscosity and microcirculatory flow is a poorly understood area of haemodynamics, especially with relevance to serious pathological conditions. Advances in microfluidics have made it possible to study the details of blood flow in the microscale, however, important issues such as the relationship between the local microstructure and local flow characteristics have not been investigated extensively. In the present study an experimental system involving simple brightfield microscopy has been successfully developed for simultaneous, time-resolved quantification of velocity fields and local aggregation of human red blood cells (RBC) in microchannels. RBCs were suspended in Dextran and phosphate buffer saline solutions for the control of aggregation. Local aggregation characteristics were investigated at bulk and local levels using statistical and edge-detection image processing techniques. A special case of aggregating flow in a microchannel, in which hematocrit gradients were present, was studied as a function of flowrate and time. The level of aggregation was found to strongly correlate with local variations in velocity in both the bulk flow and wall regions. The edge detection based analysis showed that near the side wall large aggregates are associated with regions corresponding to high local velocities and low local shear. On the contrary, in the bulk flow region large aggregates occurred in regions of low velocity and high erythrocyte concentration suggesting a combined effect of hematocrit and velocity distributions on local aggregation characteristics. The results of this study showed that using multiple methods for aggregation quantification, albeit empirical, could help towards a robust characterisation of the structural properties of the fluid.

Spatial distributions of red blood cells significantly alter local haemodynamics.

Although bulk changes in red blood cell concentration between vessels have been well characterised, local distributions are generally overlooked. Red blood cells aggregate, deform and migrate within vessels, forming heterogeneous distributions which have considerable effect on local haemodynamics. The present study reports data on the local distribution of human red blood cells in a sequentially bifurcating microchannel, representing the branching geometry of the microvasculature. Imaging methodologies with simple extrapolations are used to infer three dimensional, time-averaged velocity and haematocrit distributions under a range of flow conditions. Strong correlation between the bluntness of the velocity and haematocrit profiles in the parent branch of the geometry is observed and red blood cell aggregation has a notable effect on the observed trends. The two branches of the first bifurcation show similar characteristics in terms of the shapes of the profiles and the extent of plasma skimming, despite the difference in geometric configuration. In the second bifurcation, considerable asymmetry between the branches in the plasma skimming relationship is observed, and elucidated by considering individual haematocrit profiles. The results of the study highlight the importance of considering local haematocrit distributions in the analysis of blood flow and could lead to more accurate computational models of blood flow in microvascular networks. The experimental approaches developed in this work provide a foundation for further examining the characteristics of microhaemodynamics.

Hematocrit, viscosity and velocity distributions of aggregating and non-aggregating blood in a bifurcating microchannel.

Microscale blood flow is characterised by heterogeneous distributions of hematocrit, viscosity and velocity. In microvascular bifurcations, cells are unevenly distributed between the branches, and this effect can be amplified in subsequent branches depending on a number of parameters. We propose an approach to infer hematocrit profiles of human blood flowing through a bifurcating microchannel. The influence of aggregation, induced by the addition of Dextran 2000 to the samples, is also considered. Averaged values indicate plasma skimming, particularly in the presence of red blood cell (RBC) aggregation. Using an empirical model, the hematocrit profiles are used to estimate local relative viscosity distributions. Simulations are used to predict how the non-uniform viscosity influences the velocity profiles. Comparing these data to velocity profiles of RBCs measured using particle image velocimetry provides validation of the model. It is observed that aggregation blunts velocity profiles after a long straight section of channel. Downstream of the bifurcation, skewing of the velocity profiles is detected, which is enhanced by aggregation. The proposed methodology is capable of providing hitherto unreported information on important aspects of microscale blood rheology.

Large scale simulation of red blood cell aggregation in shear flows.

Aggregation of highly deformable red blood cells (RBCs) significantly affects the blood flow in the human circulatory system. To investigate the effect of deformation and aggregation of RBCs in blood flow, a mathematical model has been established by coupling the interaction between the fluid and the deformable solids. The model includes a three-dimensional finite volume method solver for incompressible viscous flows, the combined finite-discrete element method for computing the deformation of the RBCs, a JKR model-Johnson, Kendall and Roberts (1964-1971) (Johnson et al., 1971) to take account of the adhesion forces between different RBCs and an iterative direct-forcing immersed boundary method to couple the fluid-solid interactions. The flow of 49,512 RBCs at 45% concentration under the influence of aggregating forces was examined, improving the existing knowledge on simulating flow and structural characteristics of blood at a large scale: previous studies on the particular issue were restricted to simulating the flow of 13,000 aggregative ellipsoidal particles at a 10% concentration. The results are in excellent agreement with experimental studies. More specifically, both the experimental and the simulation results show uniform RBC distributions under high shear rates (60-100/s) whereas large aggregation structures were observed under a lower shear rate of 10/s. The statistical analysis of the simulation data also shows that the shear rate has significant influence on both the flow velocity profiles and the frequency distribution of the RBC orientation angles.

Effects of saliva on starch-thickened drinks with acidic and neutral pH.

Powdered maize starch thickeners are used to modify drink consistency in the clinical management of dysphagia. Amylase is a digestive enzyme found in saliva which breaks down starch. This action is dependent on pH, which varies in practice depending on the particular drink. This study measured the effects of human saliva on the viscosity of drinks thickened with a widely used starch-based thickener. Experiments simulated a possible clinical scenario whereby saliva enters a cup and contaminates a drink. Citric acid (E330) was added to water to produce a controlled range of pH from 3.0 to 7.0, and several commercially available drinks with naturally low pH were investigated. When saliva was added to thickened water, viscosity was reduced to less than 1% of its original value after 10-15 min. However, lowering pH systematically slowed the reduction in viscosity attributable to saliva. At pH 3.5 and below, saliva was found to have no significant effect on viscosity. The pH of drinks in this study ranged from 2.6 for Coca Cola to 6.2 for black coffee. Again, low pH slowed the effect of saliva. For many popular drinks, having pH of 3.6 or less, viscosity was not significantly affected by the addition of saliva.

Blood viscosity modelling: influence of aggregate network dynamics under transient conditions.

This paper reports on a theoretical examination of the hypothesis that red blood cell network characteristics influence the mechanical properties of the fluid. For this purpose a newly developed energy-rate based blood viscosity model, which incorporates network dynamics, was used to predict the transient behaviour of blood viscosity (steady-state results of this model have been reported in Biorheology 46 (2009), 487-508). The main network characteristic examined in the present work was the inter-aggregate branch size and its relationship to the evolving aggregates. Branch size was used to define a network integrity index that accounted for the strength of the developed network. For the development and validation of the model, experiments performed with an optical shearing microscope, with different step-changes in shear rate, were utilised, as well as viscosity measurements under similar flow conditions performed in a double wall Couette instrument. The experimental data were compared with the response of the model, which incorporated the network integrity index. The results suggest that network characteristics may influence the viscosity of blood at low shear rates and exhibit good agreement with experimental observations.

Spatial variation of blood viscosity: modelling using shear fields measured by a µPIV based technique.

The spatial characteristics of blood viscosity were investigated by combining a newly developed constitutive equation with shear deformation fields calculated from velocity measurements obtained by a µPIV based technique. Blood at physiological hematocrit levels and in the presence of aggregation was sheared in a narrow gap plate-plate geometry and the velocity and aggregation characteristics were determined from images captured using a high resolution camera. Changes in the microstructure of blood caused by aggregation were observed to affect the flow characteristics. At low shear rates, high aggregation and network formation caused the RBC motion to become essentially two-dimensional. The measured velocity fields were used to estimate the magnitude of shear which was subsequently used in conjunction with the new model to assess the spatial variation of viscosity across the flow domain. It was found that the non-uniform microstructural characteristics of blood influence its viscosity distribution accordingly. The viscosity of blood estimated in the core of the examined flow, using a zero-gradient core velocity profile assumption, was found to be significantly higher than the overall effective viscosity determined using other velocity profile assumptions.

Erythrocyte aggregation at non-steady flow conditions: a comparison of characteristics measured with electrorheology and image analysis.

In the present study electro-rheology (Contraves LS30 viscometer-based system) and optical shearing microscopy (Lincam CSS450 system and image analysis) techniques have been utilized in order to provide quantitative data on the behaviour of the microstructural properties of whole normal human blood at non-steady flow conditions. The objective of this work is to contribute towards a better understanding of red blood cell aggregation at flow conditions similar to that occurring in a circulatory system and to aid the interpretation and validation of electro-rheological data through a quantitative comparison with data acquired with optical shearing microscopy. Electro-rheology is a promising technique that has been used to provide bulk fluid properties, showing potential for basic research and diagnostic purposes, whereas optical shearing techniques offer a direct assessment of blood microstructure at a cellular level. However, little information exists in the literature regarding the relationships between electro-rheological measurements and blood microstructural characteristics. The results showed that the different non-steady flow conditions affect differently the dynamics of aggregation varying from a parabolic-decrease to an inverted S-shape curve with time. For a wide range of the non-steady flows results obtained with the two different techniques agree to a difference between 1.2 and 12%.

Coupled human erythrocyte velocity field and aggregation measurements at physiological haematocrit levels.

Simultaneous measurement of erythrocyte (RBC) velocity fields and aggregation properties has been successfully performed using an optical shearing microscope and Particle Image Velocimetry (PIV). Blood at 45% haematocrit was sheared at rates of 5.4< or =gamma < or = 252 s(-1) and imaged using a high speed camera. The images were then processed to yield aggregation indices and flow velocities. Negligible levels of aggregation were observed for gamma > or = 54.0 s(-1), while high levels of aggregation and network formation occurred for gamma < or = 11.7 s(-1). The results illustrate that the velocity measurements are dependent on the extent of RBC aggregation. High levels of network formation cause the velocities at gamma > or = 5.4 s(-1) to deviate markedly from the expected solid body rotation profile. The effect of aggregation level on the PIV accuracy was assessed by monitoring the two-dimensional (2D) correlation coefficients. Lower levels of aggregation result in poorer image correlation, from which it can be inferred that PIV accuracy is reduced. Moreover, aggregation is time-dependent, and consequently PIV accuracy may decrease during recording as the cells break up. It is therefore recommended that aggregation and its effects are taken into account in future when undertaking blood flow studies using PIV. The simplicity of the technique, which requires no lasers, filters, or special pretreatments, demonstrates the potential wide-spread applicability of the data acquisition system for accurate blood flow PIV and aggregation measurement.

An energy-rate based blood viscosity model incorporating aggregate network dynamics.

Existing time-dependent blood viscosity models that involve aggregation dynamics are mainly based on structural variables and/or viscoelastic models in order to describe the bulk mechanical properties of the fluid, but the implications of important characteristics of blood microstructure, such as the time- and flow-dependent characteristics of the red blood cell network developed due to aggregation at low shear rates, have not been thoroughly investigated. In this paper a time-dependent blood viscosity model is developed based on an energy-rate model previously proposed (Skalak et al., Biophys. J. 35 (1977), 771-781), which describes the total work needed to overcome the various forces developed between aggregated cells, including the adhesive, elastic and dissipative forces. Novel formulations of the forces acting on the fluid are developed and implemented in a volume-averaged version of the energy-rate model. The calculation of the viscosity is based on the relationship between the rate of energy changes and shear stress per unit volume of the fluid. The results show that network characteristics may significantly influence the viscosity blood at low shear rates and exhibit good agreement with experimental observations.

On the effect of microstructural changes of blood on energy dissipation in Couette flow.

Red blood cell aggregation affects the flow of blood at low shear rates; not only the behaviour of the fluid deviates from its Newtonian characteristics, but, depending on the shearing history of the flow, the non-Newtonian characteristics may be influenced. It is not clear how the time and flow-dependent characteristics of the microstructural network developed in blood affect its mechanical properties. The present study aims to improve understanding of the effect of dynamic flow conditions on microstructural characteristics and consequently on the mechanical properties of the fluid. Viscosity measurements on blood samples from healthy volunteers (H=0.45) were taken with a double-walled Couette rheometric cell, under unsteady and quasi-unsteady flow conditions. The aggregation extent index A(alpha), and the microstructural integrity index A(I) were assessed with an optical shearing system and image analysis. Results showed that energy losses in Couette geometries may depend on the structural integrity of the developed RBC network.