Francois T.H. Yu

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.

A local increase in red blood cell aggregation can trigger deep vein thrombosis: evidence based on quantitative cellular ultrasound imaging

Summary.  Background: Recurrent deep vein thrombosis (DVT) risk factors include a first idiopathic DVT, strongly suggesting the existence of undiagnosed and/or unidentified prothrombotic abnormalities. Objectives: To evaluate the impact of locally increased red blood cell (RBC) aggregation on DVT pathogenesis in a rabbit model. Methods: DVT presence, flow and aggregation were measured in situ with ultrasound. Greatly enhanced aggregation was achieved by covalent linkage of Pluronic F98 to the RBC surface; coating with Pluronic F68, which very mildly enhances aggregation, was used as a coating control. On day 1, endothelial damage and a partial stenosis were surgically created on the left femoral vein whereas the right femoral vein was not manipulated. Results: A thrombus was formed within 30 min in six out of seven left femoral veins of animals receiving a 30% volume blood exchange with F98‐coated RBC, whereas a thrombus occurred in only one out of seven veins in F68‐transfused controls. In vivo imaging using quantitative ultrasound confirmed increased aggregation in the thrombosed veins of the F98 group compared with the F68 group and the contralateral vessel. For each group, five animals were followed for 2 weeks before being killed. In F98‐transfused animals, lysis of clots occurred and the presence of chronic thrombi totally occluding the vein in three out of five animals was confirmed by histology. Conversely, in the F68 group, a single disorganized blood clot was observed in one out of five animals. Conclusions: A marked increase in RBC aggregation promotes thrombosis in rabbit femoral veins, confirming a pathophysiological role of locally altered hemorheology in the onset of DVT.

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.

Cyclic Changes in Blood Echogenicity Under Pulsatile Flow Are Frequency Dependent

Previous in vivo and in vitro studies have demonstrated that blood echogenicity varies under pulsatile flow, but such changes could not always be measured at physiological stroke rates. The apparent contradiction between these studies could be a result of the use of different ultrasound frequencies. Backscattered signals from porcine blood were measured in a pulsatile Couette flow apparatus. Cyclic changes in shear rate for stroke rates of 20 to 70 beats per minute (BPM) were applied to the Couette system, and different blood samples were analyzed (normal blood and blood with hyperaggregating erythrocytes promoted with dextran). To confirm that cyclic echogenicity variations were observable, spectral analysis was performed to verify if changes in echo-amplitude corresponded to the stroke rate applied to the flow. Echogenicity was measured with two single-element transducers at 10 and 35 MHz. At 35 MHz, cyclic variations in backscatter were observed from 20 to 70 BPM. However at 10 MHz, they were detected only at 20 BPM. For all cases except for hyperaggregating red blood cells (RBCs) at 20 BPM, the magnitude of the cyclic variations were higher at 35 MHz. We conclude that cyclic variations in RBC aggregation exist at physiological stroke rates, unlike what has been demonstrated in previous in-vitro studies at frequencies of 10 MHz. The increased sensitivity at 35 MHz to small changes in aggregate size might be the explanation for the better characterization of RBC aggregation at high stroke rates. Our results corroborate in-vivo observations of cyclic blood echogenicity variations in patients using a 30-MHz intravascular ultrasound catheter.

Increased Shear Rate Resistance And Fastest Kinetics Of Erythrocyte Aggregation In Diabetes Measured With Ultrasound

OBJECTIVE—To measure with ultrasound the increased erythrocyte aggregation (EA) kinetics and adhesion energy between erythrocytes in patients with type 2 diabetes and poor metabolic control. RESEARCH DESIGN AND METHODS—Blood samples were analyzed in a Couette rheometer at 32 MHz following shear rate reductions from 500 s−1 to residual shears of 0 (stasis), 1, 2, 10, 50, 100, and 200 s−1. The increase in EA was determined with the integrated backscatter coefficient as a function of time and shear rate. RESULTS—The time required to form aggregates was shorter in diabetic patients at shear rates below 200 s−1 (P < 0.01). Erythrocytes formed larger aggregates in diabetic patients than in control subjects (P < 0.05 at 2 to 100 s−1). CONCLUSIONS—Ultrasound can potentially noninvasively demonstrate, in vivo and in situ, the impact of local abnormal EA on arteriovenous flow disorders in diabetes.

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).

Flexible integration of high-imaging- resolution and high-power arrays for ultrasound-induced thermal strain imaging (US-TSI)

Ultrasound-induced thermal strain imaging (USTSI) for carotid artery plaque detection requires both high imaging resolution (<;100 μm) and sufficient US-induced heating to elevate the tissue temperature (~1°C to 3°C within 1 to 3 cardiac cycles) to produce a noticeable change in sound speed in the targeted tissues. Because the optimization of both imaging and heating in a monolithic array design is particularly expensive and inflexible, a new integrated approach is presented which utilizes independent ultrasound arrays to meet the requirements for this particular application. This work demonstrates a new approach in dual-array construction. A 3-D printed manifold was built to support both a high-resolution 20 MHz commercial imaging array and 6 custom heating elements operating in the 3.5 to 4 MHz range. For the application of US-TSI in carotid plaque characterization, the tissue target site is 20 to 30 mm deep, with a typical target volume of 2 mm (elevation) × 8 mm (azimuthal) × 5 mm (depth). The custom heating array performance was fully characterized for two design variants (flat and spherical apertures), and can easily deliver 30 W of total acoustic power to produce intensities greater than 15 W/cm2 in the tissue target region.

Volumetric quantification of in vitro sonothrombolysis with microbubbles using high-resolution optical coherence tomography.

Several in vitro and in vivo studies have established accelerated thrombolysis using ultrasound (US) induced microbubble (MB) cavitation. However, the mechanisms underlying MB mediated sonothrombolysis are still not completely elucidated. We performed three-dimensional (3-D) volumetric optical coherence tomography (OCT) imaging before and after the application of contrast US to thrombus. The most dramatic reduction in clot volume was observed with US + MB + recombinant tissue plasminogen activator (rt-PA). Thrombus surface erosion in this group on the side of the thrombus exposed to MB and ultrasound was evident on the OCT images. This technique may assist in clarifying the mechanisms underlying sonothrombolysis, especially regarding the importance of US transducer orientation on lytic efficacy and the effects of MB cavitation on thrombus structure.

Analysis of blood clot formation with transient elastography: similarity with sol-gel transition in agar-gelatin phantoms

Blood coagulation plays an important role in many cardiovascular diseases like atherosclerosis, heart stroke and deep vein thrombosis. The characterization of blood clot mechanical properties is fundamental in determining the appropriate treatment and for understanding the etiology of these pathologies. Intuitively, the blood coagulation can be considered as a transition from a liquid to a solid state. This behavior seems to be very close to the sol-gel transition in polymer mixture. In this paper, the validity of transient elastography to follow-up the sol-gel transition of an agar-gelatin mixture is shown. The results obtained are in good agreement with the literature and theoretical predictions. Experiments were also performed on blood pig samples in vitro. The analogy between the viscoelastic behavior of the polymer mixture and that of blood clot formation as a function of time is discussed and shown to be similar.

Low Intensity Ultrasound Mediated Liposomal Doxorubicin Delivery Using Polymer Microbubbles

Cardiotoxicity is the major dose-limiting factor in the chemotherapeutic use of doxorubicin (Dox). A delivery vehicle that can be triggered to release its payload in the tumoral microvasculature but not in healthy tissue would help improve the therapeutic window of the drug. Delivery strategies combining liposomal encapsulated Dox (LDox), microbubbles (MBs), and ultrasound (US) have been shown to improve therapeutic efficacy of LDox, but much remains to be known about the mechanisms and the US conditions that maximize cytotoxicity using this approach. In this study, we compared different US pulses in terms of drug release and acute toxicity. Drug uptake and proliferation rates using low-intensity US were measured in squamous cell carcinoma cells exposed to LDox conjugated to or coinjected with polymer MBs. The aims of this study were: (1) to compare the effects of low- and high-pressure US on Dox release kinetics; (2) to evaluate whether conjugating the liposome to the MB surface (DoxLPX) is an important factor for drug release and cytotoxicity; and (3) to determine which US parameters most inhibit cell proliferation and whether this inhibition is mediated by drug release or the MB/US interaction with cells. Low-pressure US (170 kPa) at high duty cycle (stable cavitation) released up to ∼ 70% of the encapsulated Dox from the DoxLPX, thus improving Dox bioavailability and cellular uptake and leading to a significant reduction in cell proliferation at 48 h. Flow cytometry showed that US generating stable oscillations of DoxLPX significantly increased cellular Dox uptake at 4 h after US exposure compared to LDox. Drug uptake was correlated with cytotoxicity at 48 h. Our results demonstrate that Dox-containing liposomes conjugated to polymer MBs can be triggered to release ∼ 70% of their payload using noninertial US. Following release, Dox became bioavailable to the cells and induced significantly higher cytotoxicity compared to nonreleased encapsulated drug. Our findings show promise for targeted drug delivery using this theranostic delivery platform at low US intensities.