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Hepatic Vein Transit Time of SonoVue: A Comparative Study with Levovist¹

¹ From the Imaging Sciences Department, Medical Research Council Clinical Sciences Centre (A.K.P.L., N.P., R.J.E., D.O.C., S.D.T., M.J.K.B.) and Department of Medicine A (N.P., S.D.T.), Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Rd, London W12 0HS, England; and Departments of Medicine (H.C.T., S.D.T.) and Histopathology (R.D.G.), Faculty of Medicine, Imperial College London, St Mary's Hospital, London, England. Received September 2, 2004; revision requested November 10; revision received June 16, 2005; accepted July 11; final version accepted September 1. Supported by the United Kingdom Department of Health, the British Medical Research Council, and United Kingdom National Health Service Research and Development Initiative. A.K.P.L. supported by the Kodak Scholarship, Royal College of Radiologists. Address correspondence to A.K.P.L. (e-mail: a.lim{at}imperial.ac.uk).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
Purpose: To prospectively compare transit times of Levovist and SonoVue in healthy volunteers and patients with biopsy-proved hepatitis C–related liver disease.

Materials and Methods: Institutional review board approval and informed consent were obtained. Forty patients and 25 healthy volunteers were examined. Subjects fasted, a bolus of SonoVue (0.6 mL) was injected into a cubital fossa vein, and hepatic venous time–intensity profiles were measured with spectral Doppler tracing. This was repeated with two injections of Levovist (2 g) and another injection of SonoVue. Time-intensity curves of spectral Doppler signals of right and middle hepatic veins were analyzed. A sustained signal intensity increase of 10% above baseline levels indicated hepatic vein transit time (HVTT). Carotid artery audio intensity was measured in volunteers. Analysis of variance and t tests were used for statistical analysis.

Results: Twelve patients had mild hepatitis; 18, moderate or severe hepatitis; and 10, cirrhosis. Mean HVTTs in control, mild hepatitis, moderate or severe hepatitis, and cirrhosis groups were 38.3 seconds ± 2.4 (standard error), 47.5 seconds ± 6.5, 29.5 seconds ± 10.8, and 17.6 seconds ± 5.0, respectively, with Levovist (P < .001) and 29.4 seconds ± 6.9, 27.4 seconds ± 9.3, 22.9 seconds ± 4.7, and 16.4 seconds ± 4.9, respectively, with SonoVue (P < .001). HVTT decreased as severity increased at imaging with both contrast agents. There was no significant difference in HVTT between mild and moderate hepatitis groups with SonoVue; however, there were significant differences in HVTT between all patient groups with Levovist. HVTT of SonoVue was shorter than that of Levovist in all groups (P < .001) except the cirrhosis group; in this group, HVTT of the two contrast agents was similar (P = .05). No difference was observed in mean cardiopulmonary transit time for SonoVue or Levovist (9.1 seconds ± 2.4 [standard error] and 8.4 seconds ± 2.5, respectively, P = .18).

Conclusion: HVTT was significantly shorter with SonoVue than with Levovist; there was no significant difference in cardiopulmonary transit time.

© RSNA, 2006

	INTRODUCTION

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The use of microbubble contrast agents in ultrasonography (US) has improved detection and characterization of focal liver lesions (1,2). These echo enhancers have also been used as tracers, and studies have shown that transit time through the liver is shortened in patients with cirrhosis or liver metastases compared with that in control subjects (3–8). The majority of these studies have been performed with the microbubble contrast agent Levovist (Schering, Berlin, Germany), while another study was performed with Optison (Nycomed Amersham, Buchler, Mo) (9). More stable microbubble contrast agents have since been developed, with SonoVue (Bracco, Milan, Italy) being the most widely used agent in Europe.

To our knowledge, in no study have these various agents been compared to assess whether the hepatic vein transit times (HVTTs) for each are the same. The chemical make up of newer agents is different from that of older agents, such as Levovist, which are essentially air-filled microbubble contrast agents stabilized with palmitic acid as a surfactant (1). For instance, SonoVue consists of a sulfur hexafluoride gas and a phospholipid shell (10). We hypothesized that the HVTTs of these contrast agents may differ because of their different chemical properties. Thus, the purpose of our study was to prospectively compare the transit times of Levovist and SonoVue in a cohort of patients with hepatitis C–related liver disease and healthy volunteers.

	MATERIALS AND METHODS

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The study was supported by Schering, which provided Levovist; Bracco, which provided SonoVue; and Acuson/Siemens (Mountain View, Calif), which provided a Sequoia US machine. All data and information in this article were submitted at the sole discretion of the authors, none of whom are employees of the aforementioned companies.

Study Population
The study population comprised 40 patients (25 male and 15 female patients) with biopsy-proved hepatitis C virus–related liver disease who were examnied consecutively between January 2002 and July 2003. Polymerase chain reaction test results were positive for hepatitis C RNA in all patients, indicating active viremia. The mean time from biopsy to HVTT determination was 9.3 months (median, 5 months; mode, 3 months). An additional 25 healthy volunteers (16 male and nine female volunteers) were also examined. Volunteers were approached on the basis of their age and included if they had no previous medical history of any illness at direct questioning.

All subjects consumed less than 20 g of alcohol daily, and none was taking medication regularly. All subjects provided written informed consent and were excluded if a focal liver lesion was detected on baseline US scans. This study conformed to the guidelines outlined by the 1975 Declaration of Helsinki and was approved by the ethics committee of Imperial College London at the Hammersmith Hospital, London, England.

Histologic Grading and Staging
All liver biopsy findings were interpreted by a single independent liver pathologist (R.D.G.) with more than 20 years of experience, and the samples were assessed for necroinflammation and fibrosis. They were scored according to the system described by Ishak et al (11) (modified histologic activity index). The subdivision of patients into categories of mild disease and moderate or severe disease was based on the fibrosis and necroinflammatory scoring system described by Ishak et al (11): Mild hepatitis was defined as fibrosis score of less than or equal to 2 and necroinflammatory score of less than or equal to 3. Moderate or severe hepatitis was defined as fibrosis score of 3–5 and necroinflammatory score of greater than or equal to 4. Cirrhosis was defined as a fibrosis score of 6. This subdivision was used because it corresponded to the histologic separation used in the algorithm currently recommended in the United Kingdom for the care of patients with hepatitis C–related liver disease (12). The pathologist was blinded to the findings of Doppler and microbubble-enhanced US and other clinical data.

HVTTs and Carotid Arrival Times
All patients fasted overnight or for a minimum of 6 hours before US to reduce variations in splanchnic flow that could be attributed to digestion. The method used to measure HVTT was similar to that described in previous studies (3–5). Two authors (N.P., A.K.P.L., with 7 and 3 years of experience with microbubble-enhanced US, respectively) performed all examinations.

Continuous spectral Doppler tracing of a hepatic vein was performed with the patient in quiet respiration by using the Sequoia machine. Simultaneously, a small continuous-wave US probe (Huntleigh Diagnostics, Cardiff, Wales) was placed over the carotid artery of the volunteers. Doppler signals from both the hepatic vein and the carotid artery were recorded with a standard personal computer by using the stereo audio line input. The software used to perform spectral Doppler tracing and subsequent analysis of the findings was written by an author (R.J.E.) by using Matlab software (Mathworks, Natick, Mass), as described previously (3).

A cannula (21 gauge or larger) was inserted into a median cubital vein. Baseline spectral Doppler tracing of the hepatic vein was recorded for 20 seconds before injection. The time to injection was counted down with computer software; bolus injection of 6 mL of Levovist (2 g, 300 mg/mL) or 0.6 mL of SonoVue was administered over 2 seconds and immediately followed by a rapid normal saline flush (10 mL) with a three-way tap. Data acquisition was stopped after 90 seconds, and the recorded signals were analyzed immediately.

Levovist and SonoVue were injected in each subject twice, for a total of four injections. In each subject, the right and middle hepatic veins were examined with both agents. The left hepatic vein did not yield optimal tracing because of cardiac motion interference. In the volunteers, carotid arterial arrival time was measured to investigate differences in the cardiopulmonary transit time of the two contrast agents, and the first injection of SonoVue was followed by two injections of Levovist and a final injection of SonoVue. This allowed us to assess whether there was any carry-over effect when the agents were administered in the same examination. In the patient groups, two injections of Levovist were followed consecutively by two injections of SonoVue.

Examinations were performed with real-time gray-scale imaging and spectral Doppler tracing updates (duplex mode). Gray-scale imaging was performed with relatively low acoustic power settings (mechanical index, <0.1) and without the use of tissue harmonic imaging, since our previous examinations had shown that this could disrupt the microbubbles. Duplex scanning was used to ensure that the Doppler sample gate remained over the hepatic vein during data acquisition and to check for reflux of microbubbles into a hepatic vein from the inferior vena cava.

The HVTT and the arrival time of the microbubble contrast agent in the carotid artery were defined as a sustained signal intensity increase of 10% above baseline levels. The values could be read instantly with the home-written software. When the values differed between the two injections, the lesser value was used for analysis.

Statistical Analysis
The data were distributed normally, and an analysis of variance was performed with the SPSS program (version 10.1; SPSS, Chicago, Ill) to analyze differences in the sonographic indexes and measurements obtained previously between the groups. Paired t tests were used to compare the first and last injections of SonoVue, the carotid arrival times in volunteers, and the findings with Levovist and SonoVue in all groups. A post hoc t test was used to compare observations in individual groups of patients. A P value of less than .05 indicated a significant difference.

	RESULTS

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There was an age difference between all patients and healthy volunteers: Mean age was 45.8 years ± 8.6 (standard deviation) in patients and 39.3 years ± 12.4 in volunteers (t test, P = .02). There was no difference, however, between the volunteer group and the mild hepatitis group (t test, P = .6). There was also no difference in the proportion of male or female patients in either group (² test, P = .2).

Volunteers
Figure 1 shows that no difference was observed in the mean carotid arrival times of SonoVue and Levovist (9.1 seconds ± 2.4 [standard error] and 8.4 seconds ± 2.5, respectively; P = .18). There was also no significant difference in the HVTT of SonoVue before or after injection of Levovist (r² = 0.75 [Pearson correlation coefficient], P = .87) (Fig 2). These results indicate that these two agents had similar cardiopulmonary transit times and that there was no carry-over effect between injections. In each volunteer, HVTT was shorter with SonoVue than with Levovist (P < .001) (Fig 3).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 1: Graph shows mean arrival time within the carotid arteries is the same for both contrast agents, which suggests that there is no difference in cardiopulmonary transit time of the two agents. Error bars indicate standard error (SE).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 2: Graph shows good correlation between the first and second injections of SonoVue in the volunteers, which confirms that there was no carry-over effect of Levovist when the agents were injected in the same study. Crosshairs indicate plotted values.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 3: Graph shows HVTT of SonoVue is significantly shorter than that of Levovist in the volunteers. Error bars indicate standard error (SE).

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 4: Graph shows HVTTs of both Levovist and SonoVue for all groups. In all groups (except cirrhosis), the mean HVTT is shorter with SonoVue than with Levovist. Error bars indicate standard error (SE).

	DISCUSSION

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The advent of microbubble contrast agents has provided a tracer to enable functional assessment of liver vascularity. Studies have shown that HVTT is highly sensitive for cirrhosis and can be used to predict disease severity in patients with chronic liver disease (3–5). A decreasing HVTT was observed with increasing severity of chronic liver disease. The shorter HVTT of a microbubble contrast agent in patients with diffuse liver disease was thought to be secondary to arteriovenous shunting and arterialization of capillary beds in the liver and, to a lesser degree, in the pulmonary and gastrointestinal systems (3–5). Several groups (7,8) have reproduced these studies, and their findings have concurred with previous results.

This promising functional technique represets a noninvasive method for assessing the severity of liver disease and has been used predominantly with Levovist. Newer, more robust contrast agents have since been developed; currently, the most widely used contrast agent in Europe is SonoVue. These agents have different chemical properties, with SonoVue being a more stable contrast agent than Levovist and possessing a half-life of up to 6 hours (10). This study was designed to address the issue of whether the transit times in patients with chronic liver disease were similar for differing microbubble contrast agents, particularly Levovist and SonoVue.

The results of this study show that mean HVTT was shorter with SonoVue in all groups except the cirrhosis group. There was no difference in carotid artery arrival time; this finding indicates that there was no difference in cardiopulmonary transit time between the contrast agents but that the shorter HVTT with SonoVue could be secondary to different pharmacokinetics in the liver. The results could not be attributed to carry-over effects, as there was no difference between the first and last injections of SonoVue in the control group, where SonoVue injections were interspaced with two injections of Levovist.

It is possible that HVTT occurs earlier with SonoVue because this agent is more stable and provides a greater increase in signal intensity. However, the similar cardiopulmonary transit times of SonoVue and Levovist refute this hypothesis, and there is evidence in the literature to support our hypothesis that the shorter HVTT we observed with SonoVue was due to the different kinetics of these agents in the liver. Blomley et al (16) showed that Levovist is taken up in the liver and spleen beyond the vascular phase, while Lim et al (17) demonstrated definite uptake of SonoVue in the spleen but no substantial uptake in the liver. These findings were supported by Iijima et al (18), who used phase microscopy to demonstrate the phagocystosis of Levovist, Sonazoid (GE Healthcare, Oslo, Norway), and Optison by Kuppfer cells. This work was performed by using rat hepatocytes, and it is important that this phenomenon was not observed with the contrast agents SonoVue and Imavist (Alliance Pharmaceuticals, San Diego, Calif) (18).

There was better separation between the mild hepatitis and moderate or severe hepatitis groups with Levovist than with SonoVue; however, the use of both agents revealed significant differences between these groups and the cirrhosis group. This may be because Levovist is taken up in the liver parenchyma; hence, when there is marked arteriovenous shunting bypassing the liver parenchyma—as seen with cirrhosis—both agents are equally effective and show similar transit times. Our cohort was small, and it is possible that SonoVue could facilitate the differentiation between all three groups with a larger group of patients. This needs further investigation, but the liver specificity of Levovist may offer a better and more accurate assessment of the severity of chronic liver disease.

This study had several limitations. There was a 9-month delay from biopsy to HVTT measurement in the patient group. A previous study with a larger cohort of patients with hepatitis C showed that HVTT corresponded to the fibrosis score rather than necroinflammation; thus, a 9-month delay would not affect liver fibrosis (3). It is also widely accepted that liver fibrosis is thought to progress over years rather than months (3,4,12,13). In addition, the major aim of this study was to compare HVTT between Levovist and SonoVue; hence, the delay between biopsy and the microbubble examination was a less important limitation.

There was an age difference between the volunteers and the patients; however, there was no marked age difference between the volunteers and the patients with mild hepatitis. It was difficult to find older volunteers for comparison with patients with severe hepatitis; however, this factor was less important, as we compared the HVTT of two contrast agents in the same patient.

The HVTTs of Levovist and SonoVue in the cirrhosis group were not significantly different; however, the difference bordered on statistical significance, as the P value was .05. There was little difference between the HVTTs (Fig 4), and this borderline P value may have been due to the small sample size (n = 10).

This study only demonstrated that there is no carry-over effect of Levovist on SonoVue. Whether SonoVue has a carry-over effect on Levovist was not determined; therefore, Levovist was always injected first in the patients.

In conclusion, to our knowledge, this is the first study to show that different microbubble contrast agents will yield different transit times; therefore, different contrast agents cannot be used interchangeably. The differences can likely be attributed to the different chemical properties of these agents and their pharmacokinetics in the liver. Nonetheless, HVTT measurement with microbubble contrast agents remains a promising technique for predicting the severity of liver disease noninvasively, but specific limits of normality must be determined for each agent. These limits will be important in functional examinations with microbubble contrast agents.

	ACKNOWLEDGMENTS

 
We are grateful to Sr Mary Crossey, SRN, Sr Theresa Roguin, SRN, the staff of the liver unit at St Mary's Hospital, and the staff of the gastroenterology unit at Hammersmith Hospital for help with patient recruitment. We thank all the volunteers who participated in this study.

	FOOTNOTES

 

Abbreviations: HVTT = hepatic vein transit time

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, A.K.P.L., D.O.C.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, A.K.P.L., N.P., R.D.G., H.C.T., S.D.T., M.J.K.B.; clinical studies, A.K.P.L., N.P., R.D.G., H.C.T., D.O.C.; statistical analysis, A.K.P.L., N.P., R.J.E., M.J.K.B.; and manuscript editing, A.K.P.L., N.P., R.D.G., H.C.T., D.O.C., S.D.T., M.J.K.B.

	References

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 

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This Article Abstract Figures Only Full Text (PDF) All Versions of this Article: 2401041517v1 240/1/130    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lim, A. K. P. Articles by Blomley, M. J. K. Search for Related Content PubMed PubMed Citation Articles by Lim, A. K. P. Articles by Blomley, M. J. K.