In vitro platelet activation by an echo contrast agent.

OBJECTIVE: We investigated whether an ultrasonic echo contrast agent containing microbubbles (Levovist [SH U 508A]; Schering AG, Berlin, Germany) could in routine use activate platelets. METHODS: Levovist and its main component, galactose, were mixed with separate samples of whole blood (1.5-75 mg/mL) from 5 healthy volunteers to form a 1-mL suspension sample. After in vitro exposure to ultrasound emitted from a commercial ultrasonic scanner at a pulse frequency of 3.5 MHz with a mechanical index of 1.9 and an exposure duration of 5 minutes, 5 microL of the sample was incubated for 20 minutes with the fluorescein isothiocyanate-labeled CD61 antibody, which is a platelet-specific antigen, and the phycoerythrin-labeled CD62P (P-selectin) antibody, an activation-specific antigen, both on the platelet surface. After more than 30 minutes of fixing in 1% paraformaldehyde, flow cytometric analysis was performed. RESULTS: The percentage of CD62P-expressing platelets increased according to the concentrations of Levovist and galactose, which showed almost equal effects. Ultrasound exposure did not enhance the effect except at the highest concentration of Levovist (75 mg/mL). CONCLUSIONS: In vitro, a galactose-based echo contrast agent could not activate the platelets at its routine concentration.     

Phagocytosis of ultrasound contrast agent microbubbles by Kupffer cells

Delayed parenchymal phase images of the liver more than 5 min after IV injection of ultrasound contrast agents are thought to be related to the phagocytosis of contrast agent microbubbles by macrophages. In this study, we examined whether liver-specific macrophages, Kupffer cells, phagocytosed the microbubbles and whether their elimination affected the delayed parenchymal images of the liver. Phase-contrast microscope observations showed that Kupffer cells phagocytosed various contrast agents in vitro. Among the contrast agents used, 99% of Sonazoid and Optison, and 47% of Levovist were phagocytosed, whereas only 7.3% of SonoVue and 0% of Imavist were phagocytosed. Elimination of Kupffer cells in vivo by gadolinium chloride (GdCl(3)) resulted in decreased intensity of the delayed parenchymal images with Sonazoid and Levovist, while SonoVue showed no changes compared with control. Our findings suggested that Kupffer cells phagocytosed contrast agents and they were responsible for the delayed images of contrast ultrasound in the liver.     

Correlation between microbubble contrast-enhanced color doppler sonography and immunostaining for Kupffer cells in assessing the histopathologic grade of hepatocellular carcinoma: preliminary results

PURPOSE: The aim of this study was to determine the histopathologic grades of hepatocellular carcinomas (HCCs) on the basis of the presence of Kupffer cells, using color Doppler sonography with the liver-specific microbubble contrast agent Levovist. METHODS: Color Doppler sonograms generated by stimulated acoustic emission were obtained 7 minutes after intravenous injection of 5 ml of Levovist (300 mg/dl) in patients with histopathologically confirmed HCCs. CT scans were also obtained and evaluated, and hematoxylin and eosin staining for morphologic examination and immunostaining (anti-CD68) for detecting Kupffer cells were performed for confirmation of the sonographic findings. RESULTS: Eighteen tumors had a defect in the color Doppler signal (color void) that corresponded with the baseline gray-scale image of the tumor. On histopathologic examination, these 18 tumors were all found to be either poorly or moderately differentiated HCCs with either a marked reduction in the number of or the absence of Kupffer cells. The remaining 2 tumors showed color signals. Histopathologic examination of these 2 tumors disclosed well-differentiated components within the tumors, with Kupffer cells in the tumor tissue. CONCLUSIONS: Color Doppler sonography using a liver-specific microbubble ultrasound contrast agent appears to reflect the histopathologic features of HCCs and may thus be useful for differentiating liver tumors and determining a treatment strategy.     

The influence of ultrasound frequency and gas-body composition on the contrast agent-mediated enhancement of vascular bioeffects in mouse intestine

The induction by ultrasound (US) of petechiae and hemorrhages in mouse intestine was examined with injection of gas body-based contrast agents. Production of petechiae in the intestinal wall was enhanced by contrast agents for both continuous and pulsed (10 micros pulses repeated at 1 kHz) exposure relative to a gas body-free blank. For pulsed exposure with 10 mL/kg of Albunex, apparent thresholds for peak negative pressure amplitude were 0.42 MPa at 0.4 MHz, 0.85 MPa at 1.09 MHz and 2.3 MPa at 2.4 MHz. Results at these frequencies were the same for 10-11 cycle pulses with fixed duty cycle (0.01). Thresholds for hemorrhage into the intestinal lumen were not appreciably enhanced by added Albunex, and appear to be compatible with previously reported lithotripsy data when duty factor differences are considered. The agents PESDA, Optison and Levovist had lower thresholds (for example, 1.8 MPa for Levovist) than Albunex at 2.3 MHz, and yielded more petechiae. The thresholds for petechiae induction by US with contrast agents encroach upon the exposure range relevant to diagnostic US practice.     

Endothelial cell injury and platelet aggregation induced by contrast ultrasonography in the rat hepatic sinusoid.

OBJECTIVE: To determine whether contrast ultrasonography can affect the sinusoidal cells and platelets of the liver by using ultrastructural analysis in vivo. METHODS: Fifteen Wistar rats were placed into the following 5 groups of 3 rats each: 3 control groups comprising a sham operation group, a contrast agent injection-alone group, and an ultrasound exposure-alone group; and 2 contrast agent injection with ultrasound exposure groups, split according to excision time. After a dose of an echo contrast agent (100 mg/kg of body weight) was administered through the femoral vein, the rats that received injections were subjected to ultrasound for the first minute, no ultrasound for the next 4 minutes, and then ultrasound sweep scanning for 10 seconds. The rats were perfused via the heart with cold physiologic saline containing 2% paraformaldehyde and 2.5% glutaraldehyde solution buffered with 0.1-mol/L phosphate. The livers of the rats in 4 of the groups were excised immediately. The livers of the rats in 1 of the 2 contrast agent with ultrasound exposure groups were excised by the same procedure 5 hours after they received the injections. All specimens were studied with light and electron microscopy. RESULTS: Platelet aggregation and injury to endothelial cells were more severe in the contrast agent injection and ultrasound exposure groups than in the other groups. CONCLUSIONS: Contrast ultrasonography can cause platelet aggregation and endothelial cell damage in the rat hepatic sinusoid.     

SonoVue肝脏超声造影Kupffer相一例

随着超声造影剂与造影技术的不断发展，超声造影在临床应用、诊断和治疗方面具有重要意义，尤其明显提高了对肝脏疾病的诊断率，特别是第三代造影剂的问世，如SonoVue，Sonozoid等，肝脏SonoVue超声造影表现分为三个时期：动脉期（10-30s），门脉期（30-120s），延迟期（120-180s），而肝脏Sonozoid超声造影分为四个时期：动脉期（10-30s），门脉期（30-120s），血管-Kupffer期（120-180s），Kupffer期：从注射后10 min开始；本文报告一例罕见的61岁男性在SonoVue超声造影后出现的Kupffer相，并着重探讨其出现Kupffer相的原因。     

利声显对肝肿瘤血流信号和肝实质区回声增强作用的研究

目的:评价新型利声显对不同肝肿瘤的彩色多普勒信号和肝实质区回声的增强作用。材料和方法:肝肿瘤患者共44例,其中原发性肝癌35例、继发性肝癌5例、肝血管瘤3例、肝硬化结节1例。使用HP Sonos 2500型彩超仪,控头频率2.5MHz。将利声显稀释为浓度为300mg/ml的溶液,由外周静脉注入,持续观察肿瘤彩色多普勒信号增强程度及肝脏实质区回声。结果:彩色多普勒信号增强情况为:32例原发性肝癌和1例较大胆囊转移癌明显增强,3例原发性小结节型肝癌、4例转移性结节性肝癌、2例肝血管瘤轻度增强,肝硬化结节无增强。肝实质区图像清晰度改善,于4例原发性肝癌患者的肝区内又测及新的小病灶。结论:利声显可增强肝肿瘤的血流信号,且不同肝肿瘤的增强程度有差异。它对判断肿瘤的血供情况、初步分析肿瘤的病理性质,提高超声诊断的敏感性很有益处。     

Contrast-enhanced ultrasound improves hepatic vessel visualization after orthotopic liver transplantation

BACKGROUND: We investigated whether color-coded Doppler sonography combined with an ultrasound contrast medium would improve the assessment of liver-supplying vessels after orthotopic liver transplantation. METHODS: Forty-seven patients after orthotopic liver transplantation participated. Examinations were done without and then with the ultrasound contrast medium Levovist. Visualization of the liver-supplying vessels was assessed with a scoring system. RESULTS: Visualization of the portal vein was similar without and with contrast medium. Hepatic arteries were visualized in 39 patients without contrast medium and 46 patients with contrast medium. The remaining patient showed hepatic artery thrombosis, which was confirmed angiographically. With the use of Levovist, the examination took 3.7 min rather than the usual 6.4 min. CONCLUSION: Imaging of hepatic arteries after liver transplantation improved significantly with the use of ultrasound contrast medium. These findings are important because the early detection of blood flow through the liver after transplantation affects prognosis.     

An in vitro comparison of ultrasonic contrast agents in solutions with varying air levels

The performance, in particular, the stability of ultrasound (US) contrast agents has yet to be assessed. An in vitro system has been set up to investigate the properties of ultrasonic contrast agents under different suspension conditions. This is designed to contribute to the optimal use of agents in clinical practice. In this study, the contrast agents were introduced into solutions of different oxygen concentration levels, as might be encountered in blood, and their relative performance was assessed in terms of decay in the solution environment. The partial pressures of oxygen in those solutions ranged between 1.5 and 26 kPa. Three IV and one arterial contrast agents were used: Levovist, DMP115, Quantison and Myomap. Levovist showed the highest sensitivity to oxygen concentration in the solution, and the other three proved tolerant for the above values of oxygen concentrations.     

动态灰阶超声造影在肝肿瘤诊断中的初步探索

目的：探讨动态灰阶超声造影在肝肿瘤诊断中的初步表现和作用。方法;选择6例经手术和病理证实的肝内实质性占位病变进行灰阶超声造影和动态观察。造影剂选用Levovist,浓度为400mg/ml,以团注形式经肘部浅静脉注入。结果：6例病人证实为原发性肝癌2例,肝血管瘤2例（3个）,肝脏局灶性结节性增生1例,肝脏炎性假瘤1例。除肝脏炎性假瘤外,其余病灶经造影后均有不同程度的增强。肝癌早期呈不均匀强化,消失较快;肝血管瘤增强速度较慢,呈周边强化;肝脏局灶性结节性增生亦早期增强,但维持较长时间。结论：动态灰阶超声造影对提高肝肿瘤的诊断有其实际意义。     

Definition of Contrast Enhancement Phases of the Liver Using a Perfluoro-Based Microbubble Agent,Perflubutane Microbubbles

To define the contrast enhancement phases in the liver with perflubutane microbubbles, the liver enhancement time-intensity curves were investigated in 14 healthy volunteers. The agent was injected intravenously as a bolus and the liver was imaged with an ultrasound scanner as long as 4 h after the injection. Time-intensity curves from the hepatic artery, the intrahepatic portal vein, the hepatic vein and the parenchyma of the liver were obtained from the liver ultrasound images. The arrival of the agent in the hepatic artery, the portal vein and the hepatic vein were visually distinguishable and the mean arrival times were 19.2, 24.3 and 32.2 s after the injection, respectively. The signal intensity in these vessels increased rapidly after the arrival of the contrast and gradually reverted to baseline after the peak. In contrast, within 5 min after the injection, the intensity in the parenchyma increased and reached a plateau, which persisted for at least 2 h. The contrast enhancement phases in the liver with perflubutane microbubbles could be defined as two major phases—a vascular phase, in which the vessels are enhanced between 15 s and 10 min after injection, and a Kupffer phase, in which the parenchyma is enhanced 10 min after injection. The vascular phase is divided into three subphases: the arterial phase (15 to 45 s after injection); the portal phase (45 s to 1 min after injection); and the vasculo-Kupffer phase (1 to 10 min after injection). (E-mail: hiroko-i@hyo-med.ac.jp)     

Effect of Levovist on splanchnic hemodynamics in cirrhotic patients

This study was aimed to assess the effect of Levovist on Doppler parameters of splanchnic hemodynamics. A total of 12 patients with cirrhosis and 12 healthy subjects underwent Doppler ultrasound (US) examination of the portal vein and of the hepatic, splenic and superior mesenteric arteries before, 5 to 8 and 12 to 15 min after the start of an 8-min long IV infusion of 2.5 g of Levovist. Mean velocity and mean diameter were calculated for the portal vein. Resistance index was determined for the arteries. A significant increase of resistance index was observed in the hepatic (0.80 +/- 0.07 vs. 0.71 +/- 0.06; p < 0.01) and splenic arteries (0.72 +/- 0.06 vs. 0.64 +/- 0.06; p < 0.01) 5 to 8 min after contrast agent injection in patients with cirrhosis, but not in controls. Neither portal vein diameter nor portal flow mean velocity changed during the test in both controls and cirrhotic patients. This effect might be related to a selective trapping of microbubbles in the altered hepatic and splenic microvasculature in patients with cirrhosis rather than being artefactual. It might have implications on harmonic imaging US protocols designed to image the cirrhotic liver in the early arterial phase.     

The role of late-phase pulse inversion harmonic imaging in the detection of occult hepatic metastases.

OBJECTIVE: The purpose of this study was to investigate the efficacy of late-phase pulse inversion harmonic imaging (PIHI) in detecting occult metastases and to compare the results with helical computed tomography (CT) in a group of patients whose fundamental liver sonographic results were normal. METHODS: Thirty-two patients (21 women and 11 men; age range, 20-87 years) with a known primary malignancy were enrolled in the study. The patients were evaluated with conventional sonography, unenhanced PIHI, and PIHI 3 minutes after the injection of Levovist (SH U 508A; Schering AG, Berlin, Germany). All patients also underwent triphasic helical CT within 1 week after sonography. In 1 patient, mangafodipir-enhanced magnetic resonance imaging was performed as part of the clinical workup. RESULTS: After Levovist injection, in 4 (12.5%) of 32 patients, at least 1 hypoechoic lesion was detected by PIHI; multiple lesions were shown in 1 patient. The mean diameter of newly detected lesions was 12 mm. Triphasic helical CT also showed all of the lesions that were detected by PIHI. The diagnoses were confirmed by biopsy and CT findings in 2 patients and by the typical CT and magnetic resonance imaging findings in 1 patient. For the fourth patient, the diagnosis was confirmed by follow-up and CT. Conclusions. Late-phase PIHI is comparable to helical CT for detecting occult hepatic metastases, but it protects patients from the potentially hazardous effects of radiation and iodinated contrast agents. Further series involving a larger number of patients are needed to determine its place in the evaluation of cancer staging and treatment planning.     

腹部超声检查在结节性硬化症中的应用价值

目的分析结节硬化症(TSC)的腹部超声表现,提高对该病的认识及诊断准确率。方法回顾性分析临床确诊的96例TSC患者腹部超声表现。结果 96例患者中无病变组31例,病变组65例,无病变组和病变组在性别及年龄的差异具有统计学意义(P<0.05)。病变组65例中,共64例有肾脏病变,32例有肝脏病变,其中31例肾脏合并肝脏病变。64例肾脏病变共包括血管平滑肌脂肪瘤(AML)62例、囊肿15例、肾癌1例,其中13例AML合并囊肿,1例AML合并肾癌;32例肝脏病变共包括AML 22例、血管瘤2例、囊肿13例、钙化2例,其中7例AML合并囊肿。结论腹部超声检查能够发现TSC腹部相关病变,有助于该病诊断,是其重要影像学检查方法。     

Erythrocytes, as well as microbubble contrast agents, are important factors in improving thermal and therapeutic effects of high-intensity focused ultrasound

Erythrocytes, as well as microbubble contrast agents, are important factors in improving thermal effect of high-intensity focused ultrasound (US), or HIFU, and increasing the coagulation volume produced by HIFU irradiation. In vitro experiments used human plasma with various concentrations of human erythrocytes in combination with or without Levovist. In vivo experiments used eight Japan white rabbits with three degrees of anemia. Using a 2.17-MHz transducer, HIFU was applied for 60 s, and the temperature rise and the volume of coagulation necrosis was evaluated. There was a significant correlation between the HIFU-induced temperature rise and hematocrit, with a correlation coefficient of 0.998 (p=0.0001). Although the temperature rise was smaller at low hematocrit, it was significantly increased by adding Levovist in the suspension (p<0.01). The mean volume of coagulation necrosis was significantly greater in the rabbits with higher hematocrits (p<0.01), and that in the moderate anemia group was significantly increased by using Levovist (p<0.01).     

Qualitative diagnosis of hepatocellular carcinoma by contrast enhanced ultrasonography using Coded Harmonic Angio with Levovist

We attempted to evaluate diagnosis and characterization and to access therapeutic effects in cases of hepatocellular carcinoma (HCC) by contrast enhanced ultrasound (CEUS) using Coded Harmonic Angio (CHA) with Levovist, an intravenous ultrasound contrast agent. Fifty-seven HCC foci in 39 patients, including 37 HCC foci in 28 patients before and after transcatheter arterial chemoembolization or radio frequency ablation, were examined by CEUS using the CHA mode, which is under development. This mode uses digitally encoded pulse sequences that identify and suppress nonmoving tissue signals and enhance contrast signals from Levovist in a gray-scale format. In all cases, abundant tortuous intratumoral blood flow was visualized in the early vascular phase by continuous transmission, while tumor stain was recognized by consecutive 1-to-2 second intermittent transmission. Residual tumor area after treatment was also clearly depicted by intratumoral blood flow and partial enhancement. CEUS using CHA with Levovist is likely to become important in the qualitative diagnosis of hepatic tumor and to improve the efficacy of treatment for HCC.     

The diagnostic value of levovist in Doppler imaging of visceral arteries in patients with abdominal angina before and after angioplasty

PURPOSE: To assess the diagnostic value of Levovist in the ultrasound imaging of visceral arteries in patients with clinical symptoms of abdominal angina, before and after percutaneous transluminal angioplasty (PTA). MATERIAL AND METHOD: During a 12-month period (2000/2001) five patients with visceral arterial stenoses had ultrasound examinations and a subsequent PTA procedure. Indications for ultrasound examination were abdominal angina symptoms persisting for 3-5 years, (postprandial abdominal pain, diarrhea, and vomiting). In all patients ultrasound examinations were performed using color and spectral Doppler before and after Levovist injections. Color Doppler images and maximum blood flow velocity in stenosed visceral arteries were assessed. Patients underwent control Doppler examinations with Levovist injections to assess the effect of PTA. RESULTS: In three patients conventional Doppler examination did not allow proper evaluation of visceral arteries, because of weak color and spectral Doppler signal and in two remaining patients visceral arteries were not visualized at all. In all five patients strong enhancement of color and spectral Doppler signal was observed after Levovist administration. In all these cases a hemodynamically significant stenosis was diagnosed: coeliac trunk-2 and superior mesenteric artery-3. PTA was performed successfully in these patients. In one of them ultrasound examination done before Levovist injection allowed good visualization of treated SMA and showed good PTA result. In the remaining four patients Doppler examination with the use of Levovist demonstrated visceral arteries well and confirmed successful PTA procedures. CONCLUSIONS: The use of Levovist makes the diagnostic efficiency of Doppler examinations much higher. In most cases it allows an unequivocal diagnosis of visceral artery stenosis in patients with abdominal angina symptoms. The Doppler examination with the use of Levovist is the method of choice in follow-up after PTA.     

Stimulation of Lipogenesis by Interleukin-6 and Misoprostol-Free Acid in Isolated Rat Hepatocytes

Recent work has established that Kupffer cell products, including prostaglandins, can act directly on hepatocytes to modify glucose and lipid metabolism. Additionally, prostaglandins can act on Kupffer cells to modify the expression of cytokines. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) stimulate hepatic lipogenesis following in vivo administration. To define the direct effects of these cytokines on lipogenesis in primary culture rat hepatocytes, hepatocytes were cultured in the presence of IL-6 or TNF-alpha for periods of 24--72 h. IL-6 caused an increase in hepatocyte lipogenic capacity (56% increase by 12.5 ng ml(minus sign1) IL-6 after 72 h of cytokine exposure). The increase in cellular lipogenic capacity was confirmed using (3)H2O as the radiotracer. TNF-alpha did not increase the rate of hepatocyte lipogenesis. Neither IL-6 nor TNF-alpha modified rates of lipogenesis upon acute exposure to the cytokine. Misoprostol-free acid (0.1 &mgr;M) acutely increased hepatocyte lipogenic rates by 14% in the presence of glucagon. These results demonstrate that IL-6 can act directly on hepatocytes to induce lipogenic capacity and that E-series prostaglandins can antagonize the acute inhibition of lipogenesis by glucagon. Because IL-6 is produced by Kupffer cells, and its expression is modulated by prostaglandins, the Kupffer cell is a novel target for prostaglandin therapy. Administration of prostaglandins may provide a novel strategy for pharmacologic therapy of disorders of glucose or lipid metabolism.     

Analysis of color Doppler signal intensity variation after levovist injection: a new approach to the diagnosis of thyroid nodules.

The objective of this study was to evaluate the usefulness of a galactose-based ultrasonographic contrast agent, Levovist (Schering AG, Berlin, Germany), in differentiating benign from malignant thyroid nodules by analysis of the time-intensity curves correlating the variation of the intensity signal value during the contrast transit time. Fifty-four patients scheduled for surgical removal of a nodule or the thyroid gland or both after cytologic examination were enrolled in this study; all of the nodules underwent a baseline color and power Doppler evaluation and then to a color Doppler examination after an intravenous bolus injection of Levovist. The time-intensity curves were analyzed with respect to the histologic results. Carcinomas showed a significantly earlier arrival time of Levovist than nodular hyperplastic benign nodules and adenomas (8.1 +/- 1.41 versus 19.6 +/- 2.2 and 16.1 +/- 2.8 seconds; P < .0001), although no significant difference occurred between hyperplastic benign nodules and adenomas; carcinomas and adenomas showed an earlier time to peak than hyperplastic benign nodules (14.6 +/- 1.2 and 23.1 +/- 3.8 versus 33.0 +/- 3.0 seconds; P < .0001). No significant difference was found in baseline, peak, final intensity signal, and percent variation of intensity signal among hyperplastic benign nodules, adenomas, and carcinomas. Although cytologic examination still remains the standard of reference for the presurgical diagnosis of thyroid nodules, the preliminary data of this pilot study demonstrate that the analysis of time-intensity curves after Levovist injection might provide useful, complementary, and quantitative information to differentiate benign from malignant thyroid nodules.     

Left-sided hepatic hydrothorax diagnosed by contrast-enhanced ultrasonography with intraperitoneal injection of Levovist

A 62-year-old Japanese woman was admitted to our hospital with dyspnea. Chest X-ray revealed massive pleural effusion on the left side. Contrast-enhanced ultrasonography using Levovist was performed to confirm the transdiaphragmatic passage of ascitic fluid into the pleural cavity. After injection of Levovist into the peritoneal cavity, an enhanced pulsative flow into the pleural cavity was detected. This is the first report of hepatic hydrothorax diagnosed by contrast-enhanced ultrasonography. This method is safe and useful for the diagnosis of hepatic hydrothorax, and it allows observation of the real-time movement of ascitic fluid from the peritoneal cavity to the pleural space and detection of the site of the peritoneopleural communication.