Influence of storage and buffer composition on the mechanical behavior of flowing red blood cells.

On-chip study of blood flow has emerged as a powerful tool to assess the contribution of each component of blood to its overall function. Blood has indeed many functions, from gas and nutrient transport to immune response and thermal regulation. Red blood cells play a central role therein, in particular through their specific mechanical properties, which directly influence pressure regulation, oxygen perfusion, or platelet and white cell segregation toward endothelial walls. As the bloom of in-vitro studies has led to the apparition of various storage and sample preparation protocols, we address the question of the robustness of the results involving cell mechanical behavior against this diversity. The effects of three conservation media (EDTA, citrate, and glucose-albumin-sodium-phosphate) and storage time on the red blood cell mechanical behavior are assessed under different flow conditions: cell deformability by ektacytometry, shape recovery of cells flowing out of a microfluidic constriction, and cell-flipping dynamics under shear flow. The impact of buffer solutions (phosphate-buffered saline and density-matched suspension using iodixanol/Optiprep) are also studied by investigating individual cell-flipping dynamics, relative viscosity of cell suspensions, and cell structuration under Poiseuille flow. Our results reveal that storing blood samples up to 7 days after withdrawal and suspending them in adequate density-matched buffer solutions has, in most experiments, a moderate effect on the overall mechanical response, with a possible rapid evolution in the first 3 days after sample collection.

Numerical investigations of anisotropic structures of red blood cell aggregates on ultrasonic backscattering.

Although quantitative ultrasound techniques based on the parameterization of the backscatter coefficient (BSC) have been successfully applied to blood characterization, theoretical scattering models assume blood as an isotropic scattering medium. However, the red blood cell (RBC) aggregates form anisotropic structures such as rouleaux. The present study proposes an anisotropic formulation of the effective medium theory combined with the local monodisperse approximation (EMTLMA) that considers perfectly aligned prolate-shaped aggregates. Theoretical BSC predictions were first compared with computer simulations of BSCs in a forward problem framework. Computer simulations were conducted for perfectly aligned prolate-shaped aggregates and more complex configurations with partially aligned prolate-shaped aggregates for which the size and orientation of RBC aggregates were obtained from blood optical observations. The isotropic and anisotropic EMTLMA models were then compared in an inverse problem framework to estimate blindly the structural parameters of RBC aggregates from the simulated BSCs. When considering the isotropic EMTLMA, the use of averaged BSCs over different insonification directions significantly improves the estimation of aggregate structural parameters. Overall, the anisotropic EMTLMA was found to be superior to the isotropic EMTLMA in estimating the scatterer volume distribution. These results contribute to a better interpretation of scatterer size estimates for blood characterization.

Quantifying scattering from dense media using two-dimensional impedance maps.

A better understanding of ultrasound scattering in a three-dimensional (3D) medium can provide more accurate methods for ultrasound tissue characterization. The possibility of using two-dimensional impedance maps (2DZMs) based on correlation coefficients has shown promise in the case of isotropic and sparse medium [Luchies and Oelze, J. Acoust. Soc. Am. 139, 1557-1564 (2016)]. The present study investigates the use of 2DZMs in order to quantify 3D scatterer properties of dense media from two-dimensional (2D) histological slices. Two 2DZM approaches were studied: one based on the correlation coefficient and the other based on the 2D Fourier transform of 2DZMs. Both 2DZM approaches consist in estimating the backscatter coefficient (BSC) from several 2DZMs, and then the resulting BSC was fit to the theoretical polydisperse structure factor model to yield 3D scatterer properties. Simulation studies were performed to evaluate the ability of both 2DZM approaches to quantify scattering of a 3D medium containing randomly distributed polydisperse spheres or monodisperse ellipsoids. Experimental studies were also performed using the histology photomicrographs obtained from HT29 cell pellet phantoms. Results demonstrate that the 2DZM Fourier transform-based approach was more suitable than the correlation coefficient-based approach for estimating scatterer properties when using a small number of 2DZMs.

Ultrasonic backscattering and microstructure in sheared concentrated suspensions.

Quantitative ultrasound techniques based on the parametrization of the backscatter coefficient (BSC) are used to characterize concentrated particle suspensions. Specifically, a scattering model is fit to the measured BSC and the fit parameters can provide local suspension properties. The scattering models generally assume an isotropic microstructure (i.e., spatial organization) of the scatterers, whereas the sheared concentrated suspensions can develop an anisotropic microstructure. This paper studied the influence of the shear-induced anisotropic microstructure of concentrated suspensions on the ultrasonic backscattering. Experiments were conducted on suspensions of polymethylmetacrylate spheres (5.8 µm in radius) sheared in a Couette flow device to obtain anisotropic microstructure and then mixed by hand to obtain isotropic microstructure. Experimental structure factors that are related to the spatial distribution of sphere positions were obtained by comparing the BSCs of one concentrated and one diluted suspension. Finally, Stokesian dynamics numerical simulations of sheared concentrated suspensions are used to determine the pair correlation function, which is linked to the Fourier transform of the structure factor. The experimental structure factors are found to be in good agreement with numerical simulations. The numerical simulation demonstrates that the angular-dependent BSCs and structure factors are caused by the shear-induced anisotropic microstructure within the suspension.

Estimation of polydispersity in aggregating red blood cells by quantitative ultrasound backscatter analysis.

Quantitative ultrasound techniques based on the backscatter coefficient (BSC) have been commonly used to characterize red blood cell (RBC) aggregation. Specifically, a scattering model is fitted to measured BSC and estimated parameters can provide a meaningful description of the RBC aggregates' structure (i.e., aggregate size and compactness). In most cases, scattering models assumed monodisperse RBC aggregates. This study proposes the Effective Medium Theory combined with the polydisperse Structure Factor Model (EMTSFM) to incorporate the polydispersity of aggregate size. From the measured BSC, this model allows estimating three structural parameters: the mean radius of the aggregate size distribution, the width of the distribution, and the compactness of the aggregates. Two successive experiments were conducted: a first experiment on blood sheared in a Couette flow device coupled with an ultrasonic probe, and a second experiment, on the same blood sample, sheared in a plane-plane rheometer coupled to a light microscope. Results demonstrated that the polydisperse EMTSFM provided the best fit to the BSC data when compared to the classical monodisperse models for the higher levels of aggregation at hematocrits between 10% and 40%. Fitting the polydisperse model yielded aggregate size distributions that were consistent with direct light microscope observations at low hematocrits.

Nonlinear ultrasound parameter to monitor cell death in cancer cell samples.

A scaling subtraction method was proposed to analyze the radio frequency data from cancer cell samples exposed to an anti-cancer drug and to estimate a nonlinear parameter. The nonlinear parameter was found to be well correlated (R2 = 0.62) to the percentage of dead cells in apoptosis and necrosis. The origin of the nonlinearity may be related to a change in contacts between cells, since the nonlinear parameter was well correlated to the average total coordination number of binary packings (R2 ≥ 0.77). These results suggest that the scaling subtraction method may be used to early quantify chemotherapeutic treatment efficiency.

Coherent and incoherent ultrasound backscatter from cell aggregates.

The effective medium theory (EMT) was recently developed to model the ultrasound backscatter from aggregating red blood cells [Franceschini, Metzger, and Cloutier, IEEE Trans. Ultrason. Ferroelectr. Freq. Control 58, 2668-2679 (2011)]. The EMT assumes that aggregates can be treated as homogeneous effective scatterers, which have effective properties determined by the aggregate compactness and the acoustical characteristics of the cells and the surrounding medium. In this study, the EMT is further developed to decompose the differential backscattering cross section of a single cell aggregate into coherent and incoherent components. The coherent component corresponds to the squared norm of the average scattering amplitude from the effective scatterer, and the incoherent component considers the variance of the scattering amplitude (i.e., the mean squared norm of the fluctuation of the scattering amplitude around its mean) within the effective scatterer. A theoretical expression for the incoherent component based on the structure factor is proposed and compared with another formulation based on the Gaussian direct correlation function. This theoretical improvement is assessed using computer simulations of ultrasound backscatter from aggregating cells. The consideration of the incoherent component based on the structure factor allows us to approximate the simulations satisfactorily for a product of the wavenumber times the aggregate radius krag around 2.

Structure factor model for understanding the measured backscatter coefficients from concentrated cell pellet biophantoms.

Ultrasonic backscatter coefficient (BSC) measurements were performed on K562 cell pellet biophantoms with cell concentrations ranging from 0.006 to 0.30 in the 10-42 MHz frequency bandwidth. Three scattering models, namely, the fluid-filled sphere model (FFSM), the particle model (PM), and the structure factor model (SFM), were compared for modeling the scattering from an ensemble of concentrated cells. A parameter estimation procedure was developed in order to estimate the scatterer size and relative impedance contrast that could explain the measured BSCs from all the studied cell concentrations. This procedure was applied to the BSC data from K562 cell pellet biophantoms in the 10-42 MHz frequency bandwidth and to the BSC data from Chinese hamster ovary cell pellet biophantoms in the 26-105 MHz frequency bandwidth given in Han, Abuhabsah, Blue, Sarwate, and O'Brien [J. Acoust. Soc. Am. 130, 4139-4147 (2011)]. The data fitting quality and the scatterer size estimates show that the SFM was more suitable than the PM and the FFSM for modeling the responses from concentrated cell pellet biophantoms.

Effects of Size Polydispersity and Dense Media on Quantitative Ultrasound Estimates.

Quantitative ultrasound (QUS) techniques based on the backscatter coefficient (BSC) aim to characterize the scattering properties of biological tissues. A scattering model is fit to the measured BSC, and the fitted QUS parameters can provide local tissue microstructure, namely, scatterer size and acoustic concentration. However, these techniques may fail to provide a correct description of tissue microstructure when the medium is polydisperse and/or dense. The objective of this study is to investigate the effects of scatterer size polydispersity in sparse or dense media on the QUS estimates. Four scattering models (i.e., the monodisperse and polydisperse sparse models, and the monodisperse and polydisperse concentrated models based on the structure factor) are compared to assess their accuracy and reliability in quantifying the QUS estimates. Simulations are conducted with different scatterer size distributions for sparse, moderately dense, and dense media (volume fractions of 1%, 20%, and 73%, respectively). The QUS parameters are estimated by using model-based inverse methods at different center frequencies between 8 and 50 MHz. Experimental data are also analyzed using colon adenocarcinoma HT29 cell pellet biophantoms to further validate the results obtained from simulations at the volume fraction of 73%. Our findings reveal that the choice of scattering model has a significant impact on the accuracy of QUS estimates. For sufficiently high frequencies and dense media, the polydisperse concentrated model outperforms the other models and enables more accurate quantification. Furthermore, our results contribute to advancing our understanding of the complexities associated with scatterer size polydispersity and dense media in spectral-based QUS techniques.

Quantitative ultrasound techniques for assessing thermal ablation: Measurement of the backscatter coefficient from ex vivo human liver.

BACKGROUND: Understanding the changes occurring in biological tissue during thermal ablation is at the heart of many current challenges in both therapy and medical imaging research. PURPOSE: The objective of this work is to quantitatively interpret the scattering response of human liver samples, before and after thermal ablation. We report acoustic measurements performed involving n = 21 human liver samples. Thermal ablation is achieved at temperatures between 45 and 80°C and quantification of the irreversible changes in acoustic attenuation and Backscattering Coefficient (BSC) is reported, with a particular attention to the latter. METHODS: Both attenuation coefficient and BSCs were measured in the frequency range from 10 to 52 MHz. Scans were performed before heating and after cooling down. Attenuation coefficients were calculated using spectral difference method and BSC estimated using the reference phantom method. RESULTS: Strong increases of attenuation coefficients and BSCs with heating temperature were observed. Quantitative ultrasonic parameters obtained with the polydisperse structure factor model (poly-SFM)are compared to histological observations and seen to be close to hepatocyte mean diameter (HMD). CONCLUSIONS: The results presented in this study provide a description of the impact of thermal ablation in human liver tissue on acoustic attenuation and the BSC. For the first time, quantitative agreement between the Effective Scatterer Diameter (ESD) estimated from BSC and HMD was shown, highlighting the important role of cellular network in the scattering response of the medium. This core result is an important step toward the determination of the nature of scattering sources in biological tissues.

Experimental assessment of four ultrasound scattering models for characterizing concentrated tissue-mimicking phantoms.

Tissue-mimicking phantoms with high scatterer concentrations were examined using quantitative ultrasound techniques based on four scattering models: The Gaussian model (GM), the Faran model (FM), the structure factor model (SFM), and the particle model (PM). Experiments were conducted using 10- and 17.5-MHz focused transducers on tissue-mimicking phantoms with scatterer concentrations ranging from 1% to 25%. Theoretical backscatter coefficients (BSCs) were first compared with the experimentally measured BSCs in the forward problem framework. The measured BSC versus scatterer concentration relationship was predicted satisfactorily by the SFM and the PM. The FM and the PM overestimated the BSC magnitude at actual concentrations greater than 2.5% and 10%, respectively. The SFM was the model that better matched the BSC magnitude at all the scatterer concentrations tested. Second, the four scattering models were compared in the inverse problem framework to estimate the scatterer size and concentration from the experimentally measured BSCs. The FM did not predict the concentration accurately at actual concentrations greater than 12.5%. The SFM and PM need to be associated with another quantitative parameter to differentiate between low and high concentrations. In that case, the SFM predicted the concentration satisfactorily with relative errors below 38% at actual concentrations ranging from 10% to 25%.

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.

Ultrasound characterization of red blood cell aggregation with intervening attenuating tissue-mimicking phantoms.

The analysis of the ultrasonic frequency-dependent backscatter coefficient of aggregating red blood cells reveals information about blood structural properties. The difficulty in applying this technique in vivo is due to the frequency-dependent attenuation caused by intervening tissue layers that distorts the spectral content of signals backscattered by blood. An optimization method is proposed to simultaneously estimate tissue attenuation and blood structure properties, and was termed the structure factor size and attenuation estimator (SFSAE). An ultrasound scanner equipped with a wide-band 25 MHz probe was used to insonify porcine blood sheared in both Couette and tubular flow devices. Since skin is one of the most attenuating tissue layers during in vivo scanning, four skin-mimicking phantoms with different attenuation coefficients were introduced between the transducer and the blood flow. The SFSAE gave estimates with relative errors below 25% for attenuations between 0.115 and 0.411 dBMHz and kR<2.08 (k being the wave number and R the aggregate radius). The SFSAE can be useful to examine in vivo and in situ abnormal blood conditions suspected to promote pathophysiological cardiovascular consequences.

Quantitative assessment of media concentration using the Homodyned K distribution.

The Homodyned K distribution has been used successfully as a tool in the ultrasound characterization of sparse media, where the scatterer clustering parameter α accurately discriminates between media with different numbers of scatterers per resolution cell. However, as the number of scatterers increases and the corresponding amplitude statistics become Rician, the reliability of the α estimates decreases rapidly. In the present study, we assess the usefulness of α for the characterization of both sparse and concentrated media, using simulated independent and identically distributed (i.i.d.) samples from Homodyned K distributions, ultrasound images of media with up to 68 scatterers per resolution cell and ultrasound signals acquired from particle phantoms with up to 101 scatterers per resolution cell. All parameter estimates are obtained using the XU estimator (Destrempes et al., 2013). Results suggest that the parameter α can be used to distinguish between media with up to 40 scatterers per resolution cell at 22 MHz, provided that parameter estimation can be performed on very large sample sizes (i.e., >10,000 i.i.d. samples).

Probing the Cellular Size Distribution in Cell Samples Undergoing Cell Death.

A polydisperse scattering model adapted for concentrated medium, namely the polydisperse structure factor model, was examined to explain the backscatter coefficients (BSCs) measured from packed cell samples undergoing cell death. Cell samples were scanned using high-frequency ultrasound in the 10-42 MHz bandwidth. A parameter estimation procedure was proposed to estimate the volume fraction and the relative impedance contrast that could explain the changes in BSC pattern by considering the actual change in cellular size distribution. Quantitative ultrasound parameters were estimated and related to the percentage of dead cells determined by flow cytometry. The standard deviation of scatterer size distribution extracted from the polydisperse structure factor model and the spectral intercept were found to be strongly correlated to the percentage of dead cells (r2 = 0.79 and r2 = 0.72, respectively). This study contributes to the understanding of ultrasonic scattering from cells undergoing cell death toward the monitoring of cancer therapy.

Quantitative Ultrasound in Ex Vivo Fibrotic Rabbit Livers.

Liver fibrosis is the common result of chronic liver disease. Diagnosis and grading liver fibrosis for patient management is mainly based on blood tests and hepatic puncture-biopsy, which is particularly invasive. Quantitative ultrasound (QUS) techniques provide insight into tissue microstructure and are based on the frequency-based analysis of the signals from biologic tissues. This study aims to quantify how spectral-based QUS parameters change with fibrosis grade. The changes in QUS parameters of healthy and fibrotic rabbit liver samples were investigated and were compared with the changes in liver stiffness, using shear wave elastography. Overall, the acoustic concentration was found to decrease with increasing fibrosis grade, and the effective scatterer size was found to be higher in fibrotic livers when compared with normal liver. The result of this study indicates that the combination of three QUS parameters (stiffness, effective scatterer size and acoustic concentration) provides the best classification performance, especially for classifying healthy and fibrotic livers.

Simultaneous estimation of attenuation and structure parameters of aggregated red blood cells from backscatter measurements.

The analysis of the ultrasonic frequency-dependent backscatter coefficient of aggregating red blood cells reveals information about blood structural properties. The difficulty in applying this technique in vivo is due to the frequency-dependent attenuation caused by intervening tissue layers that distorts the spectral content of backscattering properties from blood microstructures. An optimization method is proposed to simultaneously estimate tissue attenuation and blood structure factor. With in vitro experiments, the method gave satisfactory estimates with relative errors below 22% for attenuations between 0.101 and 0.317 dBcmMHz, signal-to-noise ratios>28 dB and kR<2.7 (k being the wave number and R the aggregate radius).

An optimization method for quantitative impedance tomography.

A near-field ultrasonic tomography method providing high resolution imaging for soft tissue in the reflection mode is reported. When the Born approximation is valid, the main limitation of this method is that it requires an incident pulse with infinite bandwidth, whereas the incident pulses used in practice have a limited bandwidth, which makes quantitative reconstruction impossible. The reconstructed image is qualitative in the sense that it is a band-pass filtered reconstruction of the impedance distribution. An optimization method based on the use of the geometrical information provided by the tomographic reconstruction is developed to obtain the quantitative information required. The object was approximated locally by an equivalent canonical body, on the basis of the previous global estimation. The inversion procedure is then carried out using the minimization of a cost function, which is the average over frequency of the difference between the measured field scattered by the object and the estimated field scattered by the equivalent canonical body. Assuming the object to be homogeneous by regions, the last step consists of assigning the estimated local impedance value to the region of interest. When the geometry of the real body is almost canonical, the optimization method yields accurate impedance assessments.

A 2-D anatomic breast ductal computer phantom for ultrasonic imaging.

Most breast cancers (85%) originate from the epithelium and develop first in the ductolobular structures. In screening procedures, the mammary epithelium should therefore be investigated first by the performing of an anatomically guided examination. For this purpose (mass screening, surgical guidance), we developed a two-dimensional anatomic phantom corresponding to an axial cross section of the ductolobular structures, which makes it possible to better understand the interactions between the breast composition and ultrasound. The various constitutive tissues were modeled as a random inhomogeneous continuum with density and sound speed fluctuations. Ultrasonic pulse propagation through the breast computer phantom was simulated using a finite element time domain method (the phantom can be used with other propagation codes). The simulated ductal echographic image is compared with the ductal tomographic (DT) reconstruction. The preliminary results obtained show that the DT method is more satisfactory in terms of both the contrast and the resolution.

Quantitative Characterization of Tissue Microstructure in Concentrated Cell Pellet Biophantoms Based on the Structure Factor Model.

Quantitative ultrasound (QUS) methods based on the backscatter coefficient (BSC) are typically model-based. The BSC is estimated from experiments and is fit to a model. The fit parameters are often termed QUS estimates and are used to characterize the scattering properties of the tissue under investigation. Nevertheless, for physical interpretation of QUS estimates to be accurate, the scattering model chosen must also be accurate. The goal of this work was to investigate the use of the structure factor model (SFM) to take into account coherent scattering from high volume fractions of scatterers. The study focuses on comparing the performance of two sparse models (fluid-filled sphere and Gaussian) and one concentrated model (SFM) to estimate QUS parameters from simulations and cell pellet biophantoms with a range of scatterer volume fractions. Results demonstrated the superiority of the SFM for all investigated volume fractions (i.e., from 0.006 to 0.30). In particular, the sparse models underestimated scatterer size and overestimated acoustic concentration when the volume fraction was greater than 0.12. In addition, the SFM has the ability to provide the volume fraction and the relative impedance contrast (instead of only the acoustic concentration provided by the sparse models), which could have a great benefit for tissue characterization. This study demonstrates that the SFM could prove to be an invaluable tool for QUS and could help to more accurately characterize tissue from ultrasound measurements.