Microbubble Agents Research Articles

Characterization of hepatic lesions (≤30 mm) with liver-specific contrast agents: A comparison between ultrasound and magnetic resonance imaging

PurposeImaging-based differentiation of hepatic lesions (≤30mm) between well-differentiated hepatocellular carcinomas (w-HCC) and regenerative nodules (RN) presents difficulties. The aim was to compare the diagnostic abilities to differentiate w-HCC from RN using contrast-enhanced ultrasound and magnetic resonance imaging (MRI) both with liver-specific contrast agents. Materials and methodsThis prospective study included 67 pathologically proven hepatic lesions (17.5±5.4mm, 54 w-HCCs, 13 RNs) in 56 patients with chronic hepatitis/cirrhosis (male 40, female 16; 29–79y). Hepatic-arterial/liver-specific phase enhancements were assessed quantitatively by ultrasound with perflubutane microbubble agent and MRI with gadolinium-ethoxybenzyl-diethylenetriamine with respect to the histological findings. ResultsSensitivity, specificity and accuracy of hepatic-arterial phase hyper-enhancement for w-HCC were 59.3%, 100% and 67.2% by ultrasound and 46.3%, 100% and 56.7% by MRI without significant difference. Meanwhile, those of liver-specific-phase hypo-enhancement for w-HCC were 44.4%, 100% and 55.2% by ultrasound and 87.0% (p<0.0001), 46.2% (p=0.0052) and 79.1% (p=0.0032) by MRI. Diagnostic accuracies for w-HCC by area under the receiver operating characteristic curves were higher in the hepatic-arterial phase in ultrasound (0.8316) than MRI (0.6659, p=0.0101) and similar in the liver-specific phase in ultrasound (0.7225) and MRI (0.7347, p=0.8814). ConclusionsHypervascularity is a significant feature which distinguishes w-HCC from RN, and ultrasound exerts a beneficial impact better than MRI for such characterization. However, both imaging have comparable abilities in the characterization of non-hypervascular lesions, compensating mutually for the poor sensitivity of ultrasound and the poor specificity of MRI in the liver-specific phase.

Targeted and Reversible Blood-Retinal Barrier Disruption via Focused Ultrasound and Microbubbles

The blood-retinal barrier (BRB) prevents most systemically-administered drugs from reaching the retina. This study investigated whether burst ultrasound applied with a circulating microbubble agent can disrupt the BRB, providing a noninvasive method for the targeted delivery of systemically administered drugs to the retina. To demonstrate the efficacy and reversibility of such a procedure, five overlapping targets around the optic nerve head were sonicated through the cornea and lens in 20 healthy male Sprague-Dawley rats using a 690 kHz focused ultrasound transducer. For BRB disruption, 10 ms bursts were applied at 1 Hz for 60 s with different peak rarefactional pressure amplitudes (0.81, 0.88 and 1.1 MPa). Each sonication was combined with an IV injection of a microbubble ultrasound contrast agent (Definity). To evaluate BRB disruption, an MRI contrast agent (Magnevist) was injected IV immediately after the last sonication, and serial T1-weighted MR images were acquired up to 30 minutes. MRI contrast enhancement into the vitreous humor near targeted area was observed for all tested pressure amplitudes, with more signal enhancement evident at the highest pressure amplitude. At 0.81 MPa, BRB disruption was not detected 3 h post sonication, after an additional MRI contrast injection. A day after sonication, the eyes were processed for histology of the retina. At the two lower exposure levels (0.81 and 0.88 MPa), most of the sonicated regions were indistinguishable from the control eyes, although a few tiny clusters of extravasated erythrocytes (petechaie) were observed. More severe retinal damage was observed at 1.1 MPa. These results demonstrate that focused ultrasound and microbubbles can offer a noninvasive and targeted means to transiently disrupt the BRB for ocular drug delivery.

Temporary Disruption of the Blood–Brain Barrier by Use of Ultrasound and Microbubbles: Safety and Efficacy Evaluation in Rhesus Macaques

The blood-brain barrier (BBB) prevents entry of most drugs into the brain and is a major hurdle to the use of drugs for brain tumors and other central nervous system disorders. Work in small animals has shown that ultrasound combined with an intravenously circulating microbubble agent can temporarily permeabilize the BBB. Here, we evaluated whether this targeted drug delivery method can be applied safely, reliably, and in a controlled manner on rhesus macaques using a focused ultrasound system. We identified a clear safety window during which BBB disruption could be produced without evident tissue damage, and the acoustic pressure amplitude where the probability for BBB disruption was 50% and was found to be half of the value that would produce tissue damage. Acoustic emission measurements seem promising for predicting BBB disruption and damage. In addition, we conducted repeated BBB disruption to central visual field targets over several weeks in animals trained to conduct complex visual acuity tasks. All animals recovered from each session without behavioral deficits, visual deficits, or loss in visual acuity. Together, our findings show that BBB disruption can be reliably and repeatedly produced without evident histologic or functional damage in a clinically relevant animal model using a clinical device. These results therefore support clinical testing of this noninvasive-targeted drug delivery method.

Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid microbubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin–biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1MHz pulsed ultrasound beam (0.5W/cm2) for 30s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90±6.19)%. The expression of wtp53 mRNA after transfection was (97.08±12.18)%. The cell apoptosis rate after gene therapy was (39.67±5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p<0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells.

Molecular Targeting of Ultrasonographic Contrast Agent for Detection of Head and Neck Squamous Cell Carcinoma

To investigate the feasibility of ultrasonographic (US) imaging of head and neck cancer with targeted contrast agents both in vitro and in vivo. We hypothesize that conjugation of microbubble contrast agent to tumor-specific antibodies may improve US detection of head and neck squamous cell carcinoma (HNSCC). Preclinical blinded assessment of anti-EGFR and anti-CD147 microbubble contrast agents for US imaging of HNSCC. Animal study. Immunodeficient mice. Injection of targeted microbubbles. Microbubble uptake in tumors as detected by US. In vitro assessment of anti-epidermal growth factor receptor (EGFR) and anti-CD147-targeted microbubbles in 6 head and neck cancer cell lines yielded a 6-fold improvement over normal dermal fibroblasts (P < .001). Binding of targeted agents had a positive correlation to both epidermal growth factor receptor (EGFR) (R(2) = 0.81) and CD147 (R(2) = 0.72) expression among all cell lines. In vivo imaging of flank tumors in nude mice (N = 8) yielded enhanced resolution of anti-EGFR-and anti-CD147-targeted microbubble agents over IgG control (P < .001), while dual-targeted contrast agents offered enhanced imaging over single-targeted contrast agents (P = .02 and P = .05, respectively). In a blinded in vivo assessment, targeted contrast agents increased intratumoral enhancement of flank tumors over controls. Targeted US contrast agents to both EGFR and CD147 were 100% sensitive and 87% specific in the detection of flank tumors. This preclinical study demonstrates feasibility of using molecular US to target HNSCC for contrast-enhanced imaging of HNSCC tumor in vivo.

EFSUMB Guidelines 2011: Comment on Emergent Indications and Visions

The focus of this article is the emergent and potential indications of contrast-enhanced ultrasound (CEUS). Emergent applications of CEUS techniques include extravascular and intracavitary contrast-enhanced ultrasound, quantitative assessment of microvascular circulation for tumor response assessment, and tumor characterization using dynamic contrast-enhanced ultrasound (DCE-US). Potential indications for microbubble agents include novel molecular imaging and drug and gene delivery techniques, which have been successfully tested in animal models. "Comments and Illustrations of the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) Non-Liver Guidelines 2011" which focus more on established applications are published in the same supplement to Ultraschall in der Medizin (European Journal of Ultrasound).

The kinetics of blood brain barrier permeability and targeted doxorubicin delivery into brain induced by focused ultrasound

Focused ultrasound (FUS) combined with a circulating microbubble agent is a promising strategy to non-invasively disrupt the blood–brain barrier (BBB) and could enable targeted delivery of therapeutics that normally do not leave the brain vasculature. This study investigated the kinetics of the BBB permeability using dynamic contrast-enhanced MRI (DCE-MRI) and the resulting payload of the chemotherapy agent, doxorubicin (DOX). We also investigated how the disruption and drug delivery were affected by a double sonication (DS) with two different time intervals (10 or 120min). Two locations were sonicated transcranially in one hemisphere of the brain in 20 rats using a 690kHz FUS transducer; the other hemisphere served as a control. For BBB disruption, 10ms bursts were applied at 1Hz for 60s and combined with IV injection of a microbubble ultrasound contrast agent (Definity; 10μl/kg). DOX was injected immediately after the second location was sonicated. The transfer coefficient (Ktrans) for an MRI contrast agent (Gd-DTPA) was estimated serially at 4–5 time points ranging from 30min to 7.5hrs after sonication using DCE-MRI. After a single sonication (SS), the mean Ktrans was 0.0142±0.006min−1 at 30min and was two or more orders of magnitude higher than the non-sonicated targets. It decreased exponentially as a function of time with an estimated half-life of 2.22hrs (95% confidence intervals (CI): 1.06–3.39hrs). Adding a second sonication increased Ktrans, and with a 120min interval between sonications, prolonged the duration of the BBB disruption. Mean Ktrans estimates of 0.0205 (CI: 0.016–0.025) and 0.0216 (CI: 0.013–0.030) min−1 were achieved after DS with 10 and 120min delays, respectively. The half-life of the Ktrans decay that occurred as the barrier was restored was 1.8hrs (CI: 1.20–2.41hrs) for a 10min interval between sonications and increased to 3.34hrs (CI: 0.84–5.84hrs) for a 120min interval. DOX concentrations were significantly greater than in the non-sonicated brain for all experimental groups (p<0.0001), and 1.5-fold higher for DS with a 10min interval between sonications. A linear correlation was found between the DOX concentration achieved and the Ktrans measured at 30min after sonication (R: 0.7). These data suggest that one may be able to use Gd-DTPA as a surrogate tracer to estimate DOX delivery to the brain after FUS-induced BBB disruption. The results of this study provide information needed to take into account the dynamics BBB disruption over time after FUS.

Heterogeneous Staining in the Liver Parenchyma After the Injection of Perflubutane Microbubble Contrast Agent

This study aimed to characterize the features of heterogeneous staining in the liver after injection of perflubutane microbubble agent (SonazoidTM, 0.0075 mL/kg). Digitized hepatic contrast sonograms from 906 subjects were reviewed to assess time-related changes in heterogeneous staining and the possible association between this effect and the clinical backgrounds was analyzed. Heterogeneous staining was found in seven subjects (0.77%) on 15-min phase sonograms. The staining initially appeared as hyper-enhanced circular spots in the liver 10 min or later after the agent injection. The number of spots increased gradually with unequally-spaced distribution. Although the staining pattern did not improve during the examination, there were no abnormal findings in vital signs or symptoms on the day and blood test results or sonograms on the following day. Heterogeneous staining is a side effect that impedes ultrasound examination. However, at present, the precise causes and underlying mechanisms of this event are unknown.

Quantitative Ultrasound for the Monitoring of Novel Microbubble and Ultrasound Radiosensitization

There is a need for cancer imaging to provide “real-time” information about the metabolic and cellular responses of tumours. Quantitative ultrasound techniques have recently been demonstrated to be a potential method of assessing tumour response at the cellular level. Anti-cancer treatments administered to xenograft-bearing mice consisted of radiotherapy and a novel antivascular therapy utilizing encapsulated microbubble agents in the presence of ultrasound. Radiation dose and microbubble concentrations were varied and the treatment modalities were given in combination to assess the possible enhancement of tumour cell death. Quantitative methods were used to non-invasively assess responses. Results demonstrated statistically significant changes in backscatter parameters (midband fit, spectral intercept) in tumours treated with high doses of radiotherapy or a high concentration of microbubbles. Combined treatments demonstrated further increases in ultrasound parameters. Histopathologic assessment was used and tumour cell death was found to correlate with increases in ultrasound parameters.

Emergency anticoagulation treatment for cirrhosis patients with portal vein thrombosis and acute variceal bleeding

Objective. To determine the safety and efficacy of anticoagulation treatment for portal vein thrombosis in cirrhosis patients with acute variceal bleeding, with patient eligibility determined by contrast ultrasonography findings. Materials and methods. This prospective study included 23 consecutive cirrhosis patients (63.8 ± 11.8 years old, 12 males and 11 females) with emergency admission for acute variceal bleeding with or without portal vein thrombus. Eligibility for anticoagulation treatment was determined by positive intra-thrombus enhancement on contrast ultrasonography (perflubutane microbubble agent, 0.0075 mL/kg) performed before endoscopy. Low-molecular-weight heparin was administered after hemostasis was achieved by band ligation. Repeated band ligation or injection sclerotherapy combined with argon plasma coagulation was performed for variceal disappearance. Results. Hemostasis was achieved in all 10 patients with active bleeding. Five of these patients had portal vein thrombus, and all showed positive intra-thrombus enhancement on contrast ultrasonography. Anticoagulation treatment of these five patients resulted in complete recanalization of the portal vein within 2–11 days. There were no significant differences in the number of endoscopic treatment sessions or the length of hospital stay between the groups with and without thrombosis, and no complications including rebleeding were reported. Long term, none of the patients who continued oral anticoagulation treatment had recurrence of thrombosis (4/5). Variceal recurrence occurred only in the non-thrombosis group (2/18) during the follow-up period (median: 351 days). Conclusions. Early anticoagulation treatment in cirrhosis patients with portal vein thrombosis and acute variceal bleeding may be safe, tolerated, and effective in cases with positive intra-thrombus enhancement on contrast ultrasonography.

Microbubble-enhanced high intensity focused ultrasound therapy: effect of exposure parameters on thermal lesion volume and temperature

Microbubble agents have been shown to increase therapeutic effect of HIFU (high intensity focused ultrasound). Here, the effects of treatment parameters on lesion volume and temperature are investigated. Ex vivo tissue was treated with a 2 MHz HIFU beam in absence and presence of the Artenga™ microbubbles at varying HIFU focal intensities (649-2316 W/cm2), microbubble concentrations, and exposure durations (3-10 s). The temperature was measured at 1 mm from focus using a K-type thermocouple. Thermal lesion volume was measured based on an ellipsoid model. Microbubbles increased the lesion volume and peak temperature achieved with HIFU. At the intensity of 2316 W/cm2, the lesion volume increased by 2-folds, and the peak temperature increased by 16° C with microbubbles. This effect depended on microbubble concentration, ultrasound intensity and exposure duration. Lower intensities and shorter time durations were required at higher microbubble concentrations to ablate the tissue. It was concluded that the efficacy of the HIFU therapy in combination with microbubbles can be controlled through ultrasound/microbubble exposure parameters

Stability analysis of ultrasound thick-shell contrast agents

The stability of thick shell encapsulated bubbles is studied analytically. 3-D small perturbations are introduced to the spherical oscillations of a contrast agent bubble in response to a sinusoidal acoustic field with different amplitudes of excitation. The equations of the perturbation amplitudes are derived using asymptotic expansions and linear stability analysis is then applied to the resulting differential equations. The stability of the encapsulated microbubbles to nonspherical small perturbations is examined by solving an eigenvalue problem. The approach then identifies the fastest growing perturbations which could lead to the breakup of the encapsulated microbubble or contrast agent.

Forced vibrations of a bubble in a liquid-filled elastic vessel

There is increasing demand for accurate characterization of the in vivo behavior of microbubble agents used for ultrasound imaging and therapy. This study examines bubble-vessel interaction, in particular the propagation of disturbances along the vessel wall. Finite element simulations of a 3 μm radius microbubble suspended in a viscous liquid and enclosed in a 4 μm radius elastic vessel were performed, and the results compared with existing analytical results for wave propagation in elastic liquid-filled tubes. The vessel wall was shown to have a significant effect upon the amplitude of bubble oscillation and hence acoustic radiation from it, as well as distension of the vessel wall. It was found that the most important factor was the ratio of the excitation frequency to the natural "ring" frequency of the vessel which in turn depends upon its dimensions and mechanical properties. As this ratio increases, the motion of the vessel wall becomes increasingly localized to the site of the bubble. It was also shown that the validity of the results obtained using the applied model of vessel elasticity is limited to frequencies below the ring frequency, and this should be taken into account in the development of protocols for ultrasound safety and/or therapeutic procedures.

Real‐time imaging of the lymphatic channels and sentinel lymph nodes of the stomach using contrast‐enhanced ultrasonography with Sonazoid in a porcine model

The contrast-enhanced ultrasound (CEUS)-guided method in combination with Sonazoid has not been clinically or experimentally evaluated with regard to its use for identifying sentinel lymph node (SLN) in the stomach. Therefore, we attempted to evaluate the usefulness of the CEUS-guided method with Sonazoid for imaging of the lymphatic channels and SLN of the stomach in a porcine model by comparing it with the conventional Evans blue dye-guided method. Twenty-eight 2 to 3-month-old swine weighing 17-30 kg were used in this experiment. Anesthesia was maintained with 2.0-3.0% isoflurane/O(2) inhalation. Sonazoid was injected into the intra- and sub-mucosal layers of the stomach. The intragastric or transcutaneous CEUS-guided method was used to identify the lymphatic channels and SLN of the stomach. Contrast imaging using the CEUS-guided method with Sonazoid enabled us to produce clear images of the afferent lymph vessel and SLN of the stomach until 2 h after the injection of Sonazoid. In addition, intranodal flow of the microbubble agent could be clearly identified using tissue linear harmonic images of the SLN. The SLN detection rate was not significantly different between the CEUS- and dye-guided methods. However, the Evans blue dye flowed out quickly (≈ 15 min after the injection) through the true SLN into the next LN of stomach. In conclusion, the use of the CEUS-guided method with Sonazoid might be the most useful clinical procedure for producing real-time images of the SLN of the stomach, and the linear harmonic images are also useful for evaluating intranodal structure within the SLN.

Delayed periportal enhancement: a characteristic finding on contrast ultrasound in idiopathic portal hypertension

Differentiation of idiopathic portal hypertension (IPH) from cirrhosis is not always easy because of their similar clinical features. This prospective study aimed to use contrast-enhanced ultrasound (US) in studying dynamic behavior of microbubble for characteristic enhancement features in IPH. The study had 101 subjects: 8 IPH, 47 cirrhosis, and 36 controls, and additional ten cirrhosis for data validation. Contrast-enhanced US with perflubutane microbubble agent was performed in the early-phase (0-60s) to compare hepatic enhancements among the three groups. Onset time of enhancement in the hepatic artery was not different among the groups, but that in the portal vein was longer in cirrhosis (19.9±5.2s; p=0.0014) and IPH (22.9±5.0s; p=0.0007) than in controls (16.6±3.4s). As for parenchymal enhancement, all controls showed homogeneous enhancement, while all IPH showed delayed periportal enhancement. Cirrhosis had three patterns: 14 with homogeneous enhancement, 28 with diffuse heterogeneous enhancement, and 5 with IPH-like delayed periportal enhancement. Homogeneous enhancement was more frequent in controls than in cirrhosis/IPH (p<0.0001), and delayed periportal enhancement was more frequent in IPH than in control/cirrhosis (p<0.0001). The duration of intensity difference between hypoenhancement/delayed enhancement area and surrounding parenchyma which exceeded the half of the mean value of the maximum intensity difference (8.75dB for IPH, 6.95dB for cirrhosis) was longer in IPH (15.6±6.9s) than cirrhosis (10.2±5.3s; p=0.0119). The data in cirrhosis were validated in the additional ten subjects. Delayed periportal enhancement on the sonograms based on perflubutane microbubble agent may be a characteristic of IPH.

WE‐E‐220‐02: Ultrasound and Microbubble Enhancement of Radiation Response in Tumours

Purpose: It is now appreciated that radiation not only damages the DNA inside tumour cells in vivo but may act by damaging the endothelial cells of the vasculature. In this study we tested the hypothesis that microbubble agents in vivo may be used apriori to cause endothelial cell perturbations thus causing “radiosensitization” of tumours. Materials and Methods: Human prostate cancer xenograft‐bearing mice (120 animals) were exposed to combinations of ultrasound (570kPa peak negative pressure with a 3kHz pulse repetition frequency for 750 milliseconds over 5 minutes), activated‐microbubbles (Definity) alone and in the presence of ultrasound at various concentrations, and radiation (160 kVp) (8 animals per group) at doses of 0, 2 and 8 Gy. In order to optimize treatments, studies undertaking the timing of the ultrasound and radiation treatments were carried out (40 animals) by introducing a time‐gap between ultrasound and radiation treatments ranging from 0 to 24 hours. In another cohort of animals (n=60) insonifying pressure and radiation dose were varied to investigate the effects of these parameters on treatment effect. In order to further demonstrate the efficacy of this type of combination treatment, multiple treatments combining radiation and ultrasound were also administered to tumour bearing mice over 3–4 weeks (40 animals). Treatment effects were monitored using staining of representative tumour sections with immunohistochemistry, clonogenic assays and growth delay studies. Results: Analyses indicated a synergistic increase in tumour cell kill due to vascular disruption caused by the combined therapies leading to endothelial cell apoptosis through the activation of the ceramide cell‐death pathway caused by microbubbles. The optimal time for combining the two treatments in order to maximize treatment synergy varied with radiation dose and appeared to be within 6 hours. Effects varied with radiation dose and insonifying ultrasound pressure. Multiple treatments indicated better therapeutic outcome compared to single treatments and caused tumour regression. Analyses indicated activation of ceramide‐mediated apoptotic cell death in endothelial cells leading to vascular disruption in tumours. Conclusions: This work forms the basis for ultrasound‐induced spatial targeting of radiotherapy enhancement. Results indicated a greater than 50‐fold increase in tumour cell death. This type of combination treatment has the potential to increase the efficacy of radiation and should permit lower radiation doses to be used with the same treatment effect as high doses of radiation when administered alone.

A comparison of 31P magnetic resonance spectroscopy and microbubble-enhanced ultrasound for characterizing hepatitis c-related liver disease

We compared in vivo hepatic (31) P magnetic resonance spectroscopy ((31) P MRS) and hepatic vein transit times (HVTT) using contrast-enhanced ultrasound with a microbubble agent to assess the severity of hepatitis C virus (HCV)-related liver disease. Forty-six patients with biopsy-proven HCV-related liver disease and nine healthy volunteers had (31) P MRS and HVTT performed on the same day. (31) P MR spectra were obtained at 1.5 T. Peak areas were calculated for metabolites, including phosphomonoesters (PME) and phosphodiesters (PDE). Patients also had the microbubble ultrasound contrast agent, Levovist (2 g), injected into an antecubital vein, and time-intensity Doppler ultrasound signals of the right and middle hepatic veins were measured. The HVTT was calculated as the time from injection to a sustained rise in Doppler signal 10% greater than baseline. The shortest times were used for analysis. Based on Ishak histological scoring, there were 15 patients with mild hepatitis, 20 with moderate/severe hepatitis and 11 with cirrhosis. With increasing severity of disease, the PME/PDE ratio was steadily elevated, while the HVTT showed a monotonic decrease. Both imaging modalities could separate patients with cirrhosis from the mild and moderate/severe hepatitis groups. No statistical difference was observed in the accuracy of each test to denote mild, moderate/severe hepatitis and cirrhosis (Fisher's exact test P =1.00). (31) P MRS and HVTT show much promise as noninvasive imaging tests for assessing the severity of chronic liver disease. Both are equally effective and highly sensitive in detecting cirrhosis.

Contrast-Enhanced Ultrasound With Perflubutane Microbubble Agent: Evaluation of Differentiation of Hepatocellular Carcinoma

The aim of this study was to evaluate the effectiveness of contrast-enhanced ultrasound with a perflubutane microbubble agent in the assessment of cellular differentiation of hepatocellular carcinoma (HCC). Continuous harmonic imaging with a low mechanical index (0.21-0.30) was performed 1, 5, and 10 minutes after i.v. contrast injection (0.0075 mL/kg). Tumor enhancement was evaluated by both subjective reading and objective intensity analysis based on the signal distribution in the nontumor parenchyma. Tumor vascularity was assessed with CT during hepatic arteriography. Sixty-four patients with 77 histologically proved HCCs (mean greatest dimension, 19.1 ± 5.3 mm)--six poorly differentiated HCCs, 45 moderately differentiated HCCs, and 26 well-differentiated HCCs--were enrolled in this prospective study. Among 64 hyperenhancing lesions on peak enhancement sonograms, four poorly differentiated HCCs and eight moderately differentiated HCCs exhibited washout within 1 minute. In addition to these 12 lesions, 36 lesions exhibited washout 5 minutes after injection, resulting in a total of 48 washout lesions. Fifty-four lesions exhibited washout 10 minutes after contrast injection (six poorly differentiated, 38 moderately differentiated, and 10 well-differentiated HCCs). Washout was more frequent in poorly than in moderately differentiated HCC (p = 0.0117) and well-differentiated HCC (p = 0.0003) in the 1-minute phase and was more frequent in moderately differentiated than in well-differentiated HCC in the 5-minute (p = 0.0026) and 10-minute (p = 0.0117) phases. Thirteen lesions were isoenhancing or hypoenhancing on peak enhancement sonograms (three moderately differentiated and 10 well-differentiated HCCs). Contrast-enhanced ultrasound and CT during hepatic arteriography did not differ significantly with respect to rate of detection of hyperenhancing lesions. The findings at contrast-enhanced ultrasound with the perflubutane microbubble agent may be predictive of cellular differentiation of HCC without needle biopsy.

Contrast-enhanced ultrasound for characterisation of hepatic lesions appearing non-hypervascular on CT in chronic liver diseases

The purpose of this prospective study was to elucidate the efficacy of using contrast-enhanced ultrasound to characterise focal hepatic lesions appearing non-hypervascular on contrast-enhanced CT in chronic liver diseases. The study population included 22 patients with cirrhosis or chronic hepatitis, who between them had 27 focal hepatic lesions smaller than 20 mm (mean 13.9 ± 3.4) that appeared non-hypervascular on contrast-enhanced CT. Contrast-enhanced ultrasound with perflubutane microbubble agent (Sonazoid, 0.0075 ml kg(-1)) was performed prior to ultrasound-guided needle biopsy, and intensity analysis was done for hepatic lesions in the early phase (-60 s) and late phase (600 s post injection). All seven early-phase hyperenhanced lesions were hepatocellular carcinoma (HCC). 20 lesions iso- or hypoenhanced during the early phase consisted of 11 regenerative nodules (RNs) and 9 HCCs. HCC was more frequent in early-phase hyperenhanced lesions than in iso- or hypoenhanced lesions (p=0.0108). Both late-phase hypoenhanced lesions were HCCs, whereas 25 late-phase isoenhanced lesions consisted of 11 RNs and 14 HCCs. The enhancement patterns of the 11 RNs included isoenhanced appearance in both the early and late phases in 8 lesions, and early-phase hypoenhancement combined with late-phase isoenhancement in the remaining 3. Both of these enhancement patterns (i.e. either iso-iso or hypo-iso) were found in 9 malignant lesions, 9 (75%) of the 12 well-differentiated HCCs. Hypervascularity on contrast-enhanced ultrasound with Sonazoid strongly suggested HCC regardless of non-hypervascularity on CT, and late-phase hypoenhancement was another possible finding of HCC. However, characterisation of hepatic lesions with other enhancement patterns was difficult using our technique.

Usefulness of sonazoid–ultrasonography during hepatectomy in patients with liver tumors: A preliminary study

To improve diagnostic accuracy of intraoperative ultrasonography (IOUS), we investigated the usefulness of new contrast medium of microbubble agent, Sonazoid as a preliminary study. We examined IOUS in 50 patients with liver tumors who underwent hepatectomy. Sonazoid was administrated intravenously and Kupffer-phase images of the tumor were observed before hepatectomy. Sonazoid was reinjected to observe the tumor vasculature. The tumors included hepatocellular carcinoma (HCC) in 25 patients, intrahepatic cholangiocarcinoma in 3, colorectal liver metastasis in 14, gastrointestinal stromal tumor in 1, and benign hematoma in 1. Liver tumors were clearly detected as perfusion defect in most cases. Small lesions (<1 cm), extra-capsular tumor growth, and portal vein tumor thrombus were also clearly detected on the Sonazoid-IOUS. Small occult tumors were detected in five cases. Differential diagnosis with suspicious non-tumorous lesions and benign mass was possible based on vascular findings at the early phase. In comparison with hepatectomy for HCC under conventional IOUS, the proportion of patients with positive surgical margin (0%) tended to be lower than that of the control group (P = 0.073). Sonazoid-IOUS is a promising useful tool to detect the precise tumor margin and small tumors, hence allowing curative hepatectomy or intraoperative ablation.