Effects of red blood cell aggregates dissociation on the estimation of ultrasound speckle image velocimetry

Ultrasound speckle image of blood is mainly attributed by red blood cells (RBCs) which tend to form RBC aggregates. RBC aggregates are separated into individual cells when the shear force is over a certain value. The dissociation of RBC aggregates has an influence on the performance of ultrasound speckle image velocimetry (SIV) technique in which a cross-correlation algorithm is applied to the speckle images to get the velocity field information. The present study aims to investigate the effect of the dissociation of RBC aggregates on the estimation quality of SIV technique. Ultrasound B-mode images were captured from the porcine blood circulating in a mock-up flow loop with varying flow rate. To verify the measurement performance of SIV technique, the centerline velocity measured by the SIV technique was compared with that measured by Doppler spectrograms. The dissociation of RBC aggregates was estimated by using decorrelation of speckle patterns in which the subsequent window was shifted as much as the speckle displacement to compensate decorrelation caused by in-plane loss of speckle patterns. The decorrelation of speckles is considerably increased according to shear rate. Its variations are different along the radial direction. Because the dissociation of RBC aggregates changes ultrasound speckles, the estimation quality of SIV technique is significantly correlated with the decorrelation of speckles. This degradation of measurement quality may be improved by increasing the data acquisition rate. This study would be useful for simultaneous measurement of hemodynamic and hemorheological information of blood flows using only speckle images.     

Improvement of ultrasound speckle image velocimetry using image enhancement techniques

Ultrasound-based techniques have been developed and widely used in noninvasive measurement of blood velocity. Speckle image velocimetry (SIV), which applies a cross-correlation algorithm to consecutive B-mode images of blood flow has often been employed owing to its better spatial resolution compared with conventional Doppler-based measurement techniques. The SIV technique utilizes speckles backscattered from red blood cell (RBC) aggregates as flow tracers. Hence, the intensity and size of such speckles are highly dependent on hemodynamic conditions. The grayscale intensity of speckle images varies along the radial direction of blood vessels because of the shear rate dependence of RBC aggregation. This inhomogeneous distribution of echo speckles decreases the signal-to-noise ratio (SNR) of a cross-correlation analysis and produces spurious results. In the present study, image-enhancement techniques such as contrast-limited adaptive histogram equalization (CLAHE), min/max technique, and subtraction of background image (SB) method were applied to speckle images to achieve a more accurate SIV measurement. A mechanical sector ultrasound scanner was used to obtain ultrasound speckle images from rat blood under steady and pulsatile flows. The effects of the image-enhancement techniques on SIV analysis were evaluated by comparing image intensities, velocities, and cross-correlation maps. The velocity profiles and wall shear rate (WSR) obtained from RBC suspension images were compared with the analytical solution for validation. In addition, the image-enhancement techniques were applied to in vivo measurement of blood flow in human vein. The experimental results of both in vitro and in vivo SIV measurements show that the intensity gradient in heterogeneous speckles has substantial influence on the cross-correlation analysis. The image-enhancement techniques used in this study can minimize errors encountered in ultrasound SIV measurement in which RBCs are used as flow tracers instead of exogenous contrast agents.     

Experimental ultrasound characterization of red blood cell aggregation using the structure factor size estimator

The frequency dependence of the ultrasonic backscattering coefficient (BSC) was studied to assess the level of red blood cell (RBC) aggregation. Three monoelement focused wideband transducers were used to insonify porcine blood sheared in a Couette flow from 9 to 30 MHz. A high shear rate was first applied to promote disaggregation. Different residual shear rates were then used to promote formation of RBC aggregates. The structure factor size estimator (SFSE), a second-order data reduction model based on the structure factor, was applied to the frequency-dependent BSC. Two parameters were extracted from the model to describe the level of aggregation at 6% and 40% hematocrits: W, the packing factor, and D the aggregate diameter, expressed in number of RBCs. Both parameters closely matched theoretical values for nonaggregated RBCs. W and D increased during aggregation with stabilized values modulated by the applied residual shear rate. Furthermore, parameter D during the kinetics of aggregation at 6% hematocrit under static conditions correlated with an optical RBC aggregate size estimation from microscopic images (r(2)=0.76). To conclude, the SFSE presents an interesting framework for tissue characterization of partially correlated dense tissues such as aggregated RBCs.     

Numerical simulation of the red blood cell aggregation and deformation behaviors in ultrasonic field

The objective of this paper is to propose an immersed boundary lattice Boltzmann method (IB-LBM) considering the ultrasonic effect to simulate red blood cell (RBC) aggregation and deformation in ultrasonic field. Numerical examples involving the typical streamline, normalized out-of-plane vorticity contours and vector fields in pure plasma under three different ultrasound intensities are presented. Meanwhile, the corresponding transient aggregation behavior of RBCs, with special emphasis on the detailed process of RBC deformation, is shown. The numerical results reveal that the ultrasound wave acted on the pure plasma can lead to recirculation flow, which contributes to the RBCs aggregation and deformation in microvessel. Furthermore, increasing the intensity of the ultrasound wave can significantly enhance the aggregation and deformation of the RBCs. And the formation of the RBCs aggregation leads to the fluctuated and dropped vorticity value of plasma in return.     

Modeling the frequency dependence (5-120 MHz) of ultrasound backscattering by red cell aggregates in shear flow at a normal hematocrit

The frequency dependence of the ultrasound signal backscattered by blood in shear flow was studied using a simulation model. The ultrasound backscattered signal was computed with a linear model that considers the characteristics of the ultrasound system and tissue acoustic properties. The tissue scattering properties were related to the position and shape of the red blood cells (RBCs). A 2D microrheological model simulated the RBC dynamics in a Couette shear flow system. This iterative model, described earlier [Biophys. J. 82, 1696-1710 (2002)], integrates the hydrodynamic effect of the flow, as well as adhesive and repulsive forces between RBCs. RBC aggregation was simulated at 40% hematocrit and shear rates of 0.05-2 s(-1). The RBC aggregate sizes ranged, on average, from 3.3 RBCs at 2 s(-1) to 33.5 cells at 0.05 s(-1). The ultrasound backscattered power was studied at frequencies between 5-120 MHz and insonification angles between 0-180 degrees. At frequencies below approximately 30 MHz, the ultrasound backscattered power increased as the shear rate was decreased and the size of the aggregates was raised. A totally different scattering behavior was noted above 30 MHz. Typical spectral slopes of the backscattered power (log-log scale) between 5-25 MHz equaled 3.8, whereas slopes down to 0.6 were measured at 0.05 s(-1), between 40-60 MHz. The ultrasound backscattered power was shown to be angle dependent at low frequencies (5-25 MHz). The anisotropy persisted at high frequencies (>25 MHz) for small aggregates (at 2 s(-1)). In conclusion, this study sheds some light on the blood backscattering behavior with an emphasis on the non-Rayleigh regime. Additional experimental studies may be necessary to validate the simulation results, and to fully understand the relation between the ultrasound backscattered power, level of RBC aggregation, shear rate, frequency, and insonification angle.     

静电场对红细胞悬液冻结特性的影响

本文以红细胞悬液为研究模型,通过在对其进行慢速冻结的过程中引入静电场,具体研究了静电场对降温过程中红细胞悬液冻结特性的影响。实验结果表明:静电场在一定程度上改变了冰晶的形成与生长特性,抑制了晶核的形成, 使晶枝出现不对称生长,并减缓了冰晶的生长速度,这些影响随着场强的增加而逐渐加强。在较强的静电场的影响下,冰晶明显变粗,最终成为块状,细胞也不再与冰晶分离而是完全融入粗大的冰晶之中,在冻结的末期,细胞不再受到冰晶挤压,从而减少了其所受的机械性损伤。     

A non-invasive method for the assessment of hemostasis in vivo by using dynamic light scattering

We present a new non-invasive method for assessing hemostasis in vivo. This method is based on the analysis of the movement characteristics of red blood cells (RBCs) during blood stasis condition. Stasis is intermittently induced by occlusion of arterial blood flow at the finger root. We assumed that under zero flow conditions, RBC movement is driven mostly by Brownian motion, and we characterized the RBC movement by utilizing the dynamic light scattering (DLS) technique in vivo. We found that during the stasis the RBCs diffusion coefficient in plasma decreases. We speculate that the RBC diffusion coefficient is most strongly related to endothelial and hemostatic activity. This assumption is supported by our findings that RBC movement, being expressed through the characteristics of the measured DLS signal, is correlative to the biological age and also is related to the coagulation factors. This new method can serve as a new diagnostic and research tool for the assessment of hemostasis and vascular function.     

Numerical Study on the Dynamics and Oxygen Uptake of Healthy and Malaria-Infected Red Blood Cells

Red blood cells (RBCs) are very important due to their role of oxygen transport from lungs. As the malaria parasite grows in the malaria-infected red blood cells (IRBCs), the properties of the cells change. In the present work, the oxygen uptake by RBCs and IRBCs at the pulmonary capillaries is simulated using a numerical technique based on the two-dimensional immersed interface method. The results for the oxygen uptake by a stationary single RBC have fair agreements with the previously reported results. The numerical results show that the malaria infection could significantly cause deterioration on the oxygen uptake by red blood cells. The results also suggest that the oxygen uptake by individual stationary RBC/IRBC would not be significantly affected by the neighboring cells provided the separation distance is about the dimension of the cell. Furthermore, it appears that the oxygen uptake by both RBCs and IRBCs is dominated by mass diffusion over the convection although the Peclet number is of the order of unity.     

Assessment of accuracy of the structure-factor-size-estimator method in determining red blood cell aggregate size from ultrasound spectral backscatter coefficient

A computer simulation study to produce ultrasonic backscatter coefficients (BSCs) from red blood cell (RBC) clusters is discussed. The simulation algorithm is suitable for generating non-overlapping, isotropic, and fairly identical RBC clusters. RBCs were stacked following the hexagonal close packing (HCP) structure to form a compact spherical aggregate. Such an aggregate was repeated and placed randomly under non-overlapping condition in the three-dimensional space to mimic an aggregated blood sample. BSCs were computed between 750 KHz and 200 MHz for samples of various cluster sizes at different hematocrits. Magnitudes of BSCs increased with mean aggregate sizes at low frequencies (<20 MHz). The accuracy of the structure-factor-size-estimator (SFSE) method in determining mean aggregate size and packing factor was also examined. A good correlation (R(2) ≥ 0.94) between the mean size of aggregates predicted by the SFSE and true size was found for each hematocrit. This study shows that for spherical aggregates there exists a region for each hematocrit where SFSE works most accurately. Typically, error of SFSE in estimating mean cluster size was <20% for dimensions between 14 and 17 μm at 40% hematocrit. This study suggests that the theoretical framework of SFSE is valid under the assumption of isotropic aggregates.     

Healthy and unhealthy red blood cell detection in human blood smears using neural networks

One of the most common diseases that affect human red blood cells (RBCs) is anaemia. To diagnose anaemia, the following methods are typically employed: an identification process that is based on measuring the level of haemoglobin and the classification of RBCs based on a microscopic examination in blood smears. This paper presents a proposed algorithm for detecting and counting three types of anaemia-infected red blood cells in a microscopic coloured image using circular Hough transform and morphological tools. Anaemia cells include sickle, elliptocytosis, microsite cells and cells with unknown shapes. Additionally, the resulting data from the detection process have been analysed by a prevalent data analysis technique: the neural network. The experimental results for this model have demonstrated high accuracy for analysing healthy/unhealthy cells. This algorithm has achieved a maximum detection of approximately 97.8% of all cells in 21 microscopic images. Effectiveness rates of 100%, 98%, 100%, and 99.3% have been achieved using neural networks for sickle cells, elliptocytosis cells, microsite cells and cells with unknown shapes, respectively.     

Controlled cell aggregation in a pulsed acoustic field

Cell aggregation in ultrasonic resonators can be obtained in a few seconds. Hundreds even thousands of cells can be levitated in suspension and generate 2D or 3D aggregates. Nevertheless, the aggregation rate and the 2D or 3D configurations of the resultant aggregates are very difficult to control. This work reports on a novel way of generating and controlling particle and cell aggregates using pulsed ultrasound. This technique specifically explores (in addition to the ultrasound wave, frequency and amplitude) the time of ultrasound application, i.e. the number of pulses as well as the pulse repetition frequency. We demonstrate that with pulsed ultrasound, particles and/or cells levitate in suspension, as with continuous ultrasound, and the aggregation rate can be modified in a controlled manner. By carefully tuning the number of pulses and the repetition frequency, the 3-D and 2-D configurations of the aggregates can be selectively generated. In addition, pulsed ultrasound limits transducer heating, thus allowing for higher acoustic energies than those currently employed with continuous ultrasound.     

Non-invasive assessment of static scatterer concentration in phantom body fluids using laser speckle contrast analysis technique

An adequate amount of supply of blood to the body organs is essential for the optimum survival and function of the cells. The Red Blood Cells (RBC) which are the most abundant cells of the blood transports hemoglobin which in turn carries oxygen from the lungs to the tissues. And hence its concentration in blood is an important factor. In this paper, we are presenting LAser Speckle Contrast Analysis (LASCA) as a tool for analyzing RBC concentration. Preliminary results obtained using body fluid such as blood mimicking phantoms are presented here. The technique described provides a non-contact, non-scanning and whole field method for assessing RBC concentration non-invasively.     

3D Monte Carlo simulations on the aggregate structures of a suspension composed of cubic hematite particles

In the previous study, from the viewpoint of surface modification technology, we considered a quasi-2D suspension in thermodynamic equilibrium in order to investigate the characteristics of magnetic cubic particles on a material surface. The present study has been expanded to include 3D Monte Carlo simulations of a suspension of magnetic cubic particles in order to discuss a regime change in the structures of cubic particle aggregates. We attempt to elucidate the dependence of a regime change in the aggregate structures on a variety of factors. The main results obtained here are summarised as follows. If the magnetic interaction strength is sufficiently large, closely packed clusters are formed by repeat and expansion of a cluster unit composed of eight particles, which may be the most preferred configuration as it gives rise to a minimum energy. A regime change in the internal structure of aggregates appears in a narrow range with increasing magnetic interaction strength. As the applied magnetic field strength is increased, closely packed clusters collapse and are transformed into wall-like clusters that are formed along the magnetic field direction. An increase in the volumetric fraction of particles induces a regime change from thick chain-like clusters to the formation of wall-like clusters.     

PMP/Cu泡沫材料中Cu颗粒的三维分布

为观测和分析铜掺杂聚-4-甲基-1-戊烯(PMP/Cu)低密度泡沫材料中铜颗粒的三维空间分布,采用高分辨X射线断层扫描技术,扫描PMP/Cu泡沫材料样品,对铜颗粒在聚合物泡沫中的分布进行了成像分析。经过图像处理和三维重构,获得铜颗粒在PMP聚合物泡沫中的三维立体分布图。结果分析显示:铜纳米粒子在PMP泡沫中存在团聚现象;不同尺寸的团聚物形态呈现出明显的多样化特征,小颗粒团聚物趋于球形,大颗粒团聚物趋于不规则的短木棒状,与在电子显微镜下直接观测到的结果一致。研究表明,该技术可以在不破坏样品的前提下,实现对有机聚合物泡沫材料中掺杂金属颗粒空间分布情况的直接观测。     

Time-dependent surface adhesive force and morphology of RBC measured by AFM

Atomic force microscopy (AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains, potentially providing legal medical experts useful information for forensic investigation. In this study, the time-dependent, morphological changes of red blood cells (RBC) under three different conditions (including controlled, room-temperature condition, uncontrolled, outdoor-environmental condition, and controlled, low-temperature condition) were observed by AFM, as well as the cellular viscoelasticity via force-vs-distance curve measurements. Firstly, the data indicate that substrate types have different effects on cellular morphology of RBC. RBC presented the typical biconcave shape on mica, whereas either the biconcave shape or flattened shape was evident on glass. The mean volume of RBCs on mica was significantly larger than that of cells on glass. Surprisingly, the adhesive property of RBC membrane surfaces was substrate type-independent (the adhesive forces were statistically similar on glass and mica). With time lapse, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition. Under the controlled low-temperature condition, however, the changes in cell volume occurred mainly due to the collapse of RBCs, and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC. Taken together, the AFM detections on the time-dependent, substrate type-dependent, environment (temperature/humidity)-dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in forensic medicine or investigations, e.g., estimating age of bloodstain or death time.     

Assessment of tissue viability by polarization spectroscopy

A new and versatile method for tissue viability imaging based on polarization spectroscopy of blood in superficial tissue structures such as the skin is presented in this paper. Linearly polarized light in the visible wavelength region is partly reflected directly by the skin surface and partly diffusely backscattered from the dermal tissue matrix. Most of the directly reflected light preserves its polarization state while the light returning from the deeper tissue layers is depolarized. By the use of a polarization filter positioned in front of a sensitive CCD-array, the light directly reflected from the tissue surface is blocked, while the depolarized light returning from the deeper tissue layers reaches the detector array. By separating the colour planes of the detected image, spectroscopic information about the amount of red blood cells (RBCs) in the microvascular network of the tissue under investigation can be derived. A theory that utilizes the differences in light absorption of RBCs and bloodless tissue in the red and green wavelength region forms the basis of an algorithm for displaying a colour coded map of the RBC distribution in a tissue. Using a fluid model, a linear relationship (cc. = 0.99) between RBC concentration and the output signal was demonstrated within the physiological range 0–4%. In-vivo evaluation using transepidermal application of acetylcholine by the way of iontophoresis displayed the heterogeneity pattern of the vasodilatation produced by the vasoactive agent. Applications of this novel technology are likely to be found in drug and skin care product development as well as in the assessment of skin irritation and tissue repair processes and even ultimately in a clinic case situation.     

High‐resolution Raman imaging reveals spatial location of heme oxidation sites in single red blood cells of dried smears

Raman confocal microscopy with 488 nm excitation wavelength supported with atomic force microscopy (AFM), scanning near‐field optical microscopy (SNOM) and UV–Vis spectrometry was used to investigate air‐dried erythrocytes (red blood cells, RBCs) in whole human blood smears. The central internal part of the cell was dominated by the laser‐induced O₂ dissociated oxyHb form as evidenced by the Fe²⁺ marker band appearing at 1356 cm⁻¹. The existence of a thin outer layer of hemoglobin in the periphery of RBCs was assigned to hemichrome. Evidence for hemichrome includes the oxidation state marker band appearing at 1376 cm⁻¹, the absence of FeO₂ band at 570 cm⁻¹ and a UV–Vis spectrum consistent with hemichrome. This is the first time that distributions of Fe²⁺/Fe³⁺ hemes inside the single RBC have been reported. The outer layer formation of hemichrome was additionally studied when RBCs were in contact with leucocytes and carotenoids of blood plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Depth-control method for integral imaging

We present a method for controlling the depth of three-dimensional (3D) images reconstructed by integral photography. Incoherent light is reflected from 3D objects, propagates through a lens array, and is captured as the first elemental images by a capturing device. The second elemental images of the 3D images are generated by numerical processing from the first elemental images in accordance with the desired depth. The optical reconstruction of 3D images at the desired depth by the second elemental images is confirmed experimentally.     

7Li MR measures of blood lithium--correlation with chemical analysis data

Lithium is used in the treatment and prophylaxis of manic-depressive illness. A narrow therapeutic range of lithium (0.4-1.2 meq/l) requires constant monitoring of these levels to avoid neurotoxicity and overdose. It is general practice to measure plasma lithium levels as a guide to monitor the therapy. Efforts to predict the concentration of Li at its active sites in the brain have led to the use of red blood cells (RBCs) because they are similar to neuronal cells. Thus RBC lithium is a very relevant clinical parameter for monitoring therapy and to observe the changes at intracellular levels under varying treatment conditions. A measure of both plasma and RBC lithium may be of significant value to physicians and researchers as lithium profile for RBCs correlate more closely with the brain lithium than plasma lithium. Although methods to measure lithium in blood such as atomic absorption or flame photometry exist, a complete quantitation of both plasma and RBC lithium requires a tedious physical separation of the two components prior to chemical analysis. On the other hand, lithium MR technique, via the use of shift reagents, can provide both plasma and RBC lithium in a single study. Here we have performed a correlation study of lithium results obtained from MR with the vitros dry-slide method on blood samples from rats treated with lithium. The results show a high degree of correlation between the two methods. Additionally, the MR measurements made on dilute samples of blood indicate that small blood samples with lithium concentration in the neighborhood of 0.08 meq/l can be measured with high accuracy and reproducibility needed for clinical purposes.     

Ultrasonic visualization of dynamic behavior of red blood cells in flowing blood

It is well known that the scatter of ultrasound by blood is mainly attributed to red blood cells (RBCs) and RBC aggregation. In the present review, researches of hemodynamic influence on RBC aggregation and ultrasound backscatter from blood were overviewed. A mock flow loop and a cylindrical chamber were employed to produce various blood flows, such as pulsatile, oscillatory, and rotational flow. The “black hole” (BLH), a dark hole at the tube center surrounded by bright zone in the cross sectional B-mode image and “bright collapsing ring” (BRCR) phenomena, appearance of bright ring at the periphery and collapse of it at the center during a pulsatile cycle, were observed under pulsatile flow. The combined effects of shear rate and flow acceleration on RBC aggregation were suggested as a possible mechanism for these phenomena. The stroke volume-dependence of the “bright ring” phenomenon under oscillatory flow could also be explained by flow acceleration. The enveloped echo images from rotational flow in a compact blood chamber showed the spatial and temporal variations of RBC aggregation, which varied with the mammalian species. In the stenotic model, it was found that the echogenic variation increased locally at a distance of three tube diameters downstream from the stenosis during decelerating period, which was proposed to be mainly due to flow turbulence. The similar ‘bright ring’ was also observed fromin vivo human carotid artery in harmonic imaging.