Radiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Forsberg, F.
Right arrow Articles by Shi, W. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Forsberg, F.
Right arrow Articles by Shi, W. T.
(Radiology. 1999;210:125-132.)
© RSNA, 1999

Experimental Studies

Tissue-Specific US Contrast Agent for Evaluation of Hepatic and Splenic Parenchyma

Flemming Forsberg, PhD1, Barry B. Goldberg, MD1, Ji-Bin Liu, MD1, Daniel A. Merton, RD, MS1, Nandkumar M. Rawool, MD1 and William T. Shi, PhD1

1 Department of Radiology, Division of Ultrasound, Thomas Jefferson University, Suite 763J, Main Bldg, 132 S 10th St, Philadelphia, PA 19107.


   Abstract
TOP
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
PURPOSE: To evaluate the recently developed ultrasonographic (US) contrast agent SHU 563A, which is specifically taken up by the reticuloendothelial system (RES).

MATERIALS AND METHODS: Color Doppler imaging (CDI) was performed in a gel phantom, with SHU 563A microbubbles in stationary suspension. CDI was performed in vivo in five woodchucks with natural hepatomas and in 12 rabbits before and after intravenous bolus injections of SHU 563A (0.16–0.48 mL/kg). After a 15–135-minute delay, the liver and spleen were scanned again, and the image findings were compared with pathologic analysis results.

RESULTS: Phantom CDI demonstrated a random mosaic color pattern in spite of the lack of flow. This phenomenon, which is associated with bubble rupture, is termed induced acoustic emission. In vivo, delayed imaging demonstrated acoustic emission signals in normal parenchyma, whereas no mosaic color was seen in regions lacking reticuloendothelial cells (eg, tumors). Four of 12 VX-2 tumors detected with pathologic analysis were detected with US alone; the remaining eight tumors were detected by using US with contrast agent (100%, P = .0078). Nine of 20 hepatomas were detected at baseline US, whereas 17 were detected after administration of SHU 563A (P = .0215). Acoustic emission enabled detection of hepatic tumors as small as 3 mm in diameter.

CONCLUSION: CDI with SHU 563A demonstrates a random mosaic color pattern, even without flow. The characteristic appearance of acoustic emission signals provides a distinctive method of visualizing normal hepatic tissues and substantially improves the detectability of hepatic tumors.

Index terms: Liver neoplasms, 761.323 • Liver, US, 761.12983, 761.12988 • Spleen, US, 775.12983, 775.12988 • Ultrasound (US), contrast media, 761.12988 • Ultrasound (US), Doppler studies, 761.12983, 761.12988 • Ultrasound (US), experimental, 761.12983, 761.12984, 761.12988


   Introduction
TOP
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
During the past decade, a number of ultrasonographic (US) contrast agents that consist of encapsulated gas microbubbles have been developed (1–3). Such agents are capable of enhancing Doppler US signal intensities and, in some cases, gray-scale echogenicity. US contrast agents are now available commercially in the United States, Europe, and elsewhere. The first such agent, Albunex (Molecular Biosystems, San Diego, Calif), has been shown to help improve echocardiographic examinations (4) but has limited applications in other areas of the body. The second agent, the galactose-based Levovist (Schering, Berlin, Germany), which is now available in a number of European countries, has been shown to enhance US Doppler signal intensity in vessels throughout the body (5,6). Other US contrast agents are currently in various stages of evaluation in both animals and humans (7–12). All of these agents can circulate for several minutes after intravenous injection; this allows sufficient time to examine specific anatomic areas of interest. However, to date, very few of these agents, and none that contain gas microbubbles, to our knowledge, have shown characteristics of being taken up by specific organs.

SHU 563A (Sonovist; Schering) is a recently developed US contrast agent that has been shown in animal studies to produce vascular enhancement after intravenous injection. In addition, it has been determined that SHU 563A is taken up (ie, phagocytosed) over time by the cells of the reticuloendothelial system (RES) in the liver and spleen (13). The purpose of this study was to evaluate this contrast agent in vitro and in vivo. In the latter experiments, we assessed the effectiveness of SHU 563A in improving the US depiction of normal parenchyma in the spleen and liver and in the detection of induced and naturally occurring liver tumors in two animal models.


   MATERIALS AND METHODS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
SHU 563A consists of air microbubbles that are approximately 1–2 µm in diameter (all bubbles are less than 7 µm) and have a biodegradable, 100-nm-thick shell composed of polybutyl-2-cyanoacrylate (13, p 170). This composition results in an agent that is stable enough to avoid dissolution in the bloodstream yet is thin and elastic enough to oscillate in an impending sound field. After intravenous injection of SHU 563A, two phases of enhancement occur. In the initial (ie, vascular) phase, the addition of microbubbles into the bloodstream produces increased signal intensities in both gray-scale and Doppler modes (color as well as spectral). This enhancement enables improved evaluation of blood flow in the arteries and veins of the systemic circulation for several minutes (14). In the second (ie, delayed) phase, the agent, by being phagocytosed by the cells of the RES, is removed from the bloodstream over a period of at least 15 minutes. In this phase, color Doppler imaging (CDI) can be used to help identify normal hepatic or splenic tissue–that is, the tissue that contains the SHU 563A microbubbles.

For the in vitro study, a gel phantom that consisted of Knox gelatin mixed with water and 1.25 mL of SHU 563A (total volume, 125 mL) was constructed and cast in a square mold. This resulted in a high concentration of microbubbles that were evenly distributed in stationary suspension within the gelatin block. In the in vivo experiments, five healthy rabbits (mean weight, 3.7 kg) were studied initially. Subsequently, seven rabbits with VX-2 liver tumors and five woodchucks (mean weight in both groups of animals, 3.6 kg) with naturally occurring hepatocellular carcinomas were included in these experiments to assess the potential of US with SHU 563A to depict hepatic tumors.

The first tumor model investigated was that of VX-2 carcinoma in rabbits. This cell line does not spread by natural means to other rabbits or humans and has been grown in many tissues in the rabbit (8,15). After injecting 0.5 mL of 3 x 106 VX-2 tumor cells (Biomeasure, Hopkinton, Mass) into the liver, an avascular mass develops in approximately 2 weeks. The second tumor model studied was that of naturally occurring hepatocellular carcinoma in woodchucks. In the wild, 70% of these animals are chronically infected with a hepatitis virus that is nontransferable to other rodents or humans. Twenty-five percent of these infected woodchucks develop hepatomas (16). Infected animals can be identified by monitoring their serum liver enzymes for the presence of woodchuck hepatitis surface antigen (16). Because there is no direct relationship between liver function and tumor development, the supplier (Marmotech, Illion, NY) performs an initial US examination in these animals before selecting those for contrast agent–enhanced studies. The results of the supplier's US study is not communicated to investigators to avoid bias.

A catheter was placed in the animals' jugular veins to provide access for injection of the contrast agent and the anesthetic. Initial sedation was induced with an intramuscular injection of 0.65 mg/kg of a mixture of ketamine hydrochloride (Ketaset; Aveco, Fort Dodge, Iowa) and xylazine hydrochloride (Gemini; Rugby Laboratory, Rockville Center, NY) and was maintained during the procedures with periodic intravenous injections of ketamine hydrochloride. The multiple injections of SHU 563A administered varied in dose from 0.5 to 1.2 mL (equivalent to 0.16–0.48 mL/kg) per injection. Each injection was followed by a 5-mL saline flush.

A Gateway US scanner (Diasonics, Santa Clara, Calif) or an HDI 3000 US unit (Advanced Technology Laboratories, Bothell, Wash), both with high-resolution, 5–10 MHz linear-array transducers, was used to obtain gray-scale and color flow measurements before and immediately after injection as well as after a 15–135-minute delay. US was performed by two experienced sonographers (D.A.M., N.M.R.), who kept all imaging parameters constant before and after contrast agent administration. The effect of varying the acoustic output power was studied. The pressure was estimated by using the mechanical index displayed on the screen. The mechanical index, which is defined as the peak rarefactional pressure (in megapascals) divided by the square root of the imaging frequency (in megahertz) (17), provides an indirect measure of the acoustic pressure and was varied between 0.1 and 1.1. At completion of the experiments, the animals were sacrificed by using humane methods.

Gross pathologic analysis of the liver was performed in the animals with tumors. After excision, the livers were scanned to establish the orientation that best matched the US imaging planes. Later, the livers were sectioned (by J.B.L.), and the location and size of the masses were noted. Hence, the results of pathologic analysis were compared directly with the tumor sizes and positions determined by using US before and after contrast agent administration. In cases where the correlation between pathologic and US findings was not obvious, two independent observers (J.B.L., F.F.) reviewed the results of both studies and established concurrence.

Statistical analyses were performed by using the McNemar test for correlated proportions. Owing to the small number of animals, the exact binomial probabilities were used in the McNemar test (18, p 345). All of the described experiments were approved by the animal use and care committee at Thomas Jefferson University, Philadelphia, Pa, and conducted in a humane manner with the guidance of a veterinarian.


   RESULTS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Initial gray-scale images in the phantom showed homogeneous echogenicity in the anterior portion, with a decrease in signal intensity posteriorly due to the high concentration of bubbles (Fig 1a). Subsequent CDI demonstrated a random mosaic color pattern in spite of the lack of flow in the gel block (Fig 1b). This phenomenon, termed "induced acoustic emission," is caused by the bubble rupture that results from the increase in output pressure in the CDI mode relative to the gray-scale mode (14,19). Notice that this increased pressure is still within the approved diagnostic range. As the energy in the acoustic field comes in contact with the agent, the microbubbles oscillate and finally collapse. During the collapse, energy is released in the form of a localized, transient, broadband signal that is detectable with an autocorrelator (ie, in CDI mode) and is depicted as a characteristic region of random pseudocolor (ie, not associated with blood flow). Afterward, a clear anechoic defect that corresponds to the area of bubble destruction can be seen with gray-scale imaging (Fig 1c).



View larger version (89K):
[in this window]
[in a new window]
  		Figure 1a. Gel phantom with stationary SHU 563A microbubbles. (a) Initial gray-scale image in the phantom. (b) Color Doppler image shows acoustic emission. (c) Gray-scale image obtained after CDI-induced acoustic emission demonstrates a hypoechoic defect (arrows) due to bubble rupture.

 


View larger version (63K):
[in this window]
[in a new window]
  		Figure 1b. Gel phantom with stationary SHU 563A microbubbles. (a) Initial gray-scale image in the phantom. (b) Color Doppler image shows acoustic emission. (c) Gray-scale image obtained after CDI-induced acoustic emission demonstrates a hypoechoic defect (arrows) due to bubble rupture.

 


View larger version (112K):
[in this window]
[in a new window]
  		Figure 1c. Gel phantom with stationary SHU 563A microbubbles. (a) Initial gray-scale image in the phantom. (b) Color Doppler image shows acoustic emission. (c) Gray-scale image obtained after CDI-induced acoustic emission demonstrates a hypoechoic defect (arrows) due to bubble rupture.

 
As with other US contrast agents, after the injection of SHU 563A in vivo, there was initial enhancement of the Doppler and gray-scale signal intensities. The effect of the contrast agent lasted an average of 5–8 minutes in this phase. The duration of vascular enhancement increased with increasing dose, similar to what has been reported with other agents (2,3). In the tumor models, imaging performed immediately after contrast agent administration did not reveal any new lesions. Hence, all of the results described in the following text refer to delayed acoustic emission imaging.

CDI of the liver and spleen in the healthy rabbits during the second phase of enhancement—that is, with imaging delayed 15–135 minutes—produced similar results; acoustic emission associated with uptake of the agent in the RES was demonstrated. With US stimulation, the microbubbles in the macrophages in the spleen collapsed; this resulted in random Doppler shifts or acoustic emission signals that appeared as regions of pseudocolor (Fig 2). However, these acoustic emission signals are not associated with any flow. The spleen shown in Figure 2 was imaged with a stand-off pad ex vivo—that is, after surgical removal of the organ.



View larger version (126K):
[in this window]
[in a new window]
  		Figure 2a. Ex vivo images in rabbit spleen after intravenous injection of 0.2 mL/kg SHU 563A. (a) Gray-scale image shows uniform echogenicity. (b) Color Doppler image shows acoustic emission.

 


View larger version (55K):
[in this window]
[in a new window]
  		Figure 2b. Ex vivo images in rabbit spleen after intravenous injection of 0.2 mL/kg SHU 563A. (a) Gray-scale image shows uniform echogenicity. (b) Color Doppler image shows acoustic emission.

 
Wherever the color box was positioned over the normal liver or spleen, the mosaic pattern associated with acoustic emission filled the entire box. When the kidney was imaged in the same plane as the liver by using CDI, blood flow signals were seen in the renal hilum, whereas acoustic emission signals were demonstrated in the liver only (Fig 3a). Notice that acoustic emission signals can be differentiated from true blood flow with CDI by the characteristic mosaic color pattern, whereas acoustic emission signals and blood flow have identical appearances at power Doppler US (Fig 3b) owing to the lack of directivity. Thus, acoustic emission effects are better appreciated with CDI than with power Doppler US.



View larger version (94K):
[in this window]
[in a new window]
  		Figure 3a. Images in normal rabbit liver and kidney 75 minutes after injection of 0.2 mL/kg SHU 563A. (a) Color Doppler image shows acoustic emission signals from within the liver (L), whereas blood flow is seen from within the renal hilum (H). (b) Power Doppler image of the same area as in a. Notice that acoustic emission signals can be differentiated from true blood flow with CDI, whereas power Doppler imaging demonstrates the blood flow and acoustic emission with identical appearances.

 


View larger version (80K):
[in this window]
[in a new window]
  		Figure 3b. Images in normal rabbit liver and kidney 75 minutes after injection of 0.2 mL/kg SHU 563A. (a) Color Doppler image shows acoustic emission signals from within the liver (L), whereas blood flow is seen from within the renal hilum (H). (b) Power Doppler image of the same area as in a. Notice that acoustic emission signals can be differentiated from true blood flow with CDI, whereas power Doppler imaging demonstrates the blood flow and acoustic emission with identical appearances.

 
Adequate visualization of bubble rupture depended on the acoustic output pressure (Fig 4). Typically, a mechanical index of greater than or equal to 0.6 was required to achieve a sufficient rate of bubble collapse, whereas only limited acoustic emission occurred with lower mechanical index values, and no acoustic emission occurred with a mechanical index of less than 0.4. In larger livers, the acoustic emission effect was best seen in the more anterior regions. However, adjustment of the color box and depth of focus did enable complete evaluation of the liver, with the caveat that the largest depth imaged in these studies was 5 cm.



View larger version (82K):
[in this window]
[in a new window]
  		Figure 4a. Color Doppler images in rabbit liver approximately 75 minutes after injection of 0.2 mL/kg SHU 563A. (a) Limited acoustic emission is seen with a mechanical index of 0.1 because the acoustic power level is insufficient to cause bubble rupture. (b) Increasing the mechanical index to 0.6 results in an acoustic emission, and the associated color display appears.

 


View larger version (70K):
[in this window]
[in a new window]
  		Figure 4b. Color Doppler images in rabbit liver approximately 75 minutes after injection of 0.2 mL/kg SHU 563A. (a) Limited acoustic emission is seen with a mechanical index of 0.1 because the acoustic power level is insufficient to cause bubble rupture. (b) Increasing the mechanical index to 0.6 results in an acoustic emission, and the associated color display appears.

 
An example of the focal zone adjustment required to delineate a naturally occurring hepatoma is shown in Figure 5, where the tumor is visualized as a colorfree area within the acoustic emission color display and thus is distinguishable from the normal hepatic parenchyma depicted by the colored areas. In some instances, incomplete destruction of the RES by the hepatoma resulted in some color pixels being displayed, but the predominance of the tumorous tissue made its recognition easy compared with the recognition of the normal tissue, which was completely filled with color (Fig 6).



View larger version (96K):
[in this window]
[in a new window]
  		Figure 5a. Color Doppler images show the effects of the transmit focal zone location on acoustic emission in woodchuck liver. (a) With a 12-mm-deep focal zone, acoustic emission signals in the anterior portion of the normal liver parenchyma demarcate the anterior border of a hepatoma (arrowheads). (b) With the color box and focal zone placed deeper (ie, at 20 mm), acoustic emission signals occur in the posterior region of the liver. The posterior border of the tumor (arrowheads) is delineated.

 


View larger version (92K):
[in this window]
[in a new window]
  		Figure 5b. Color Doppler images show the effects of the transmit focal zone location on acoustic emission in woodchuck liver. (a) With a 12-mm-deep focal zone, acoustic emission signals in the anterior portion of the normal liver parenchyma demarcate the anterior border of a hepatoma (arrowheads). (b) With the color box and focal zone placed deeper (ie, at 20 mm), acoustic emission signals occur in the posterior region of the liver. The posterior border of the tumor (arrowheads) is delineated.

 


View larger version (129K):
[in this window]
[in a new window]
  		Figure 6a. (a) Delayed color Doppler image obtained after injection of 0.16 mL/kg SHU 563A shows acoustic emission signals from within a woodchuck hepatic tumor. The tumor is surrounded by normal liver tissue eliciting acoustic emission. Additional acoustic emission signals (arrow) are present from within the tumor. While this may represent contrast-enhanced blood flow, (b) pathologic specimen confirms a small amount of normal liver tissue (arrow) within the tumor.

 


View larger version (131K):
[in this window]
[in a new window]
  		Figure 6b. (a) Delayed color Doppler image obtained after injection of 0.16 mL/kg SHU 563A shows acoustic emission signals from within a woodchuck hepatic tumor. The tumor is surrounded by normal liver tissue eliciting acoustic emission. Additional acoustic emission signals (arrow) are present from within the tumor. While this may represent contrast-enhanced blood flow, (b) pathologic specimen confirms a small amount of normal liver tissue (arrow) within the tumor.

 
In the rabbits, the VX-2 tumors were also seen as colorfree regions, and it was possible to separate individual tumors with 1 mm of normal tissue between them (Fig 7a), as confirmed by using pathologic analysis (Fig 7b). Although other images demonstrated acoustic emission signals in the hypoechoic region posterior to the two tumors, these images did not show the 1-mm strip of normal tissue as well as the image in Figure 7a. Furthermore, tumors as small as 4 x 3 mm were detected after the administration of the contrast agent (Fig 8). Initial imaging revealed a solitary 16 x 10-mm tumor (Fig 8a), but after injection of the contrast agent, the acoustic emission delineated a small (4 x 3-mm) satellite lesion nearby, albeit in a slightly different scanning plane (Fig 8b).



View larger version (152K):
[in this window]
[in a new window]
  		Figure 7a. (a) Color Doppler image of VX-2 liver tumors in a rabbit approximately 20 minutes after injection of 0.2 mL/kg SHU 563A. Acoustic emission signals clearly delineate two individual tumors (t) separated by a 1-mm strip of normal liver tissue. (b) Pathologic specimen confirms the two tumors (t).

 


View larger version (118K):
[in this window]
[in a new window]
  		Figure 7b. (a) Color Doppler image of VX-2 liver tumors in a rabbit approximately 20 minutes after injection of 0.2 mL/kg SHU 563A. Acoustic emission signals clearly delineate two individual tumors (t) separated by a 1-mm strip of normal liver tissue. (b) Pathologic specimen confirms the two tumors (t).

 


View larger version (148K):
[in this window]
[in a new window]
  		Figure 8a. (a,b) Color Doppler images of VX-2 tumors in a rabbit. (a) Color Doppler image obtained before contrast agent administration suggests a large solitary tumor (T, arrows) with poorly defined margins. (b) Delayed color Doppler image obtained 20 minutes after injection of 0.2 mL/kg SHU 563A shows the original tumor (T), as well as a second unsuspected area (t) that does not contain acoustic emission signals, which indicates the presence of an additional lesion—a small (4 x 3-mm) VX-2 tumor. (c) Pathologic specimen confirms the large tumor (T) and the small tumor (curved arrow) detected only after contrast agent administration. The photograph of the pathologic specimen was rotated 180° to correspond to b.

 


View larger version (142K):
[in this window]
[in a new window]
  		Figure 8b. (a,b) Color Doppler images of VX-2 tumors in a rabbit. (a) Color Doppler image obtained before contrast agent administration suggests a large solitary tumor (T, arrows) with poorly defined margins. (b) Delayed color Doppler image obtained 20 minutes after injection of 0.2 mL/kg SHU 563A shows the original tumor (T), as well as a second unsuspected area (t) that does not contain acoustic emission signals, which indicates the presence of an additional lesion—a small (4 x 3-mm) VX-2 tumor. (c) Pathologic specimen confirms the large tumor (T) and the small tumor (curved arrow) detected only after contrast agent administration. The photograph of the pathologic specimen was rotated 180° to correspond to b.

 


View larger version (138K):
[in this window]
[in a new window]
  		Figure 8c. (a,b) Color Doppler images of VX-2 tumors in a rabbit. (a) Color Doppler image obtained before contrast agent administration suggests a large solitary tumor (T, arrows) with poorly defined margins. (b) Delayed color Doppler image obtained 20 minutes after injection of 0.2 mL/kg SHU 563A shows the original tumor (T), as well as a second unsuspected area (t) that does not contain acoustic emission signals, which indicates the presence of an additional lesion—a small (4 x 3-mm) VX-2 tumor. (c) Pathologic specimen confirms the large tumor (T) and the small tumor (curved arrow) detected only after contrast agent administration. The photograph of the pathologic specimen was rotated 180° to correspond to b.

 
Of the 12 rabbit VX-2 tumors found at pathologic analysis, two were greater than 20 mm in diameter, two were 10–20 mm in diameter, and the remaining eight were 2–10 mm in diameter (Table 1). US with and without the contrast agent enabled detection of all four lesions larger than 10 mm. However, none of the 2–10-mm tumors were seen at baseline US—that is, with US alone—but all eight were identified after the administration of SHU 563A. Thus, an overall increase in sensitivity from 33% (four of 12 lesions) to 100% was achieved with contrast agent administration. The improvement in tumor detection achieved by using US with contrast agent relative to US alone was statistically significant (P = .0078). Although tumor implantation was successful in all seven rabbits, US alone enabled the detection of lesions in only four rabbits, while the addition of SHU 563A resulted in tumor detection in all seven rabbits. This equates to a sensitivity of 57% (four of seven rabbits) with US imaging, which increased to 100% with the addition of the contrast agent. Here, sensitivity refers to the ability to correctly identify a rabbit with tumors. Likewise, US without contrast agent enabled the identification of none of the four rabbits with two or more tumors, whereas US with contrast agent enabled the detection of multiple masses in all four animals.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Hepatic VX-2 Tumor Sizes in Rabbits Determined by Using US Alone, US with Acoustic Emission, and Pathologic Analysis
 
The hepatocellular carcinomas in woodchucks were generally more difficult to detect. US imaging performed before contrast agent administration enabled the detection of seven of the 10 lesions greater than 10 mm in diameter (Table 2). This included one tumor that was initially assigned to the largest size range (>20 mm) but after contrast agent administration was correctly found to belong in the 10–20-mm lesion group. The administration of contrast agent resulted in all 10 tumors larger than 10 mm being detected and assigned to the correct size range. However, at least one tumor was correctly detected in all woodchucks on the baseline US images. Contrast agent administration enabled the detection of seven 2–10-mm-diameter hepatomas, which is five more than the number of lesions detected by using US alone. However, three lesions seen at pathologic analysis were not visualized at US with contrast agent. Thus, the sensitivity increased from 20% (two of 10 lesions) with US alone to 70% (seven of 10 lesions) after contrast agent administration for tumors 2–10 mm in diameter. Overall sensitivity increased from 45% (nine of 20 lesions) with US alone to 85% (17 of 20 lesions) with US and contrast agent. The administration of the contrast agent resulted in a statistically significant improvement in tumor detection (P = .0215).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Hepatocellular Carcinoma Sizes in Woodchucks Determined by Using US Alone, US with Acoustic Emission, and Pathologic Analysis
 

   DISCUSSION
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
A number of capabilities of SHU 563A have been identified, including (a) enhancement of standard color and power Doppler images, (b) enhancement of gray-scale images, and (c) acoustic emission imaging characteristics. The most novel aspect of SHU 563A, however, is its affinity for the RES (Figs 2–8). Over time, this agent is taken up by the Kupffer cells in the liver and spleen. Microbubbles start accumulating almost instantly and gradually build up over many minutes, and the effects last for at least 2 hours. When using Doppler imaging (color as well as power), the increased acoustic energy relative to that with gray-scale imaging causes the microbubbles to oscillate and collapse, which results in localized random transient phase shifts. These shifts, in turn, are interpreted incorrectly by the autocorrelator as random Doppler shifts, which appear as a mosaic of colors in the regions that contain SHU 563A microbubbles. The phantom results demonstrated that the acoustic emission signals are caused by bubble rupture (Fig 1); this has been confirmed by initial reports from the manufacturer of SHU 563A (13,19).

The concept of contrast agent microbubble rupture caused by impinging acoustic pressure is not unique to SHU 563A. Other researchers (20,21) have reported a marked increase in contrast enhancement on the initial US image where the bubble concentration is higher (ie, before bubble destruction). This phenomenon has even led to a new contrast agent imaging mode, transient response imaging, being proposed (20). In transient response imaging, reduced frame rates of 5 Hz and lower are used to reduce bubble rupture and improve the effects of the contrast agent—that is, increase enhancement.

The acoustic emission color display format, however, is distinctive. The acoustic emission signals cannot be mistaken for either the color displayed when the contrast agent is in the vascular phase (ie, within blood vessels) or the flash artifact caused by tissue motion. However, distinguishing the flash artifact from acoustic emission signals in the power Doppler mode can be difficult, and therefore the acoustic emission signal is easier to visualize in the CDI mode (Fig 3). In fact, acoustic emission constitutes a unique use of CDI; all previous uses have been for the detection of motion, principally flow within blood vessels but also ureteric jets and cardiac tissue motion (22,23). Acoustic emission, as shown in our in vitro study, does not rely on motion but rather on the location of SHU 563A microbubbles. Finally, as shown in Figure 4, acoustic emission signals do not have the color blooming artifact associated with vascular contrast studies (24).

Recently, two other tissue-specific US contrast agents have been described in the literature (25,26). These efforts, which focused on contrast agents that adhere to thrombi, are still in the early stages. On the other hand, phase I trials on SHU 563A have been completed in Europe. The ability to produce acoustic emission signals with other contrast agents has been described in preliminary form in two instances (27,28); this indicates that this ability may not be unique to SHU 563A.

The attenuation seen in the phantom study (Fig 1) is not a problem in vivo, because the concentration used is much lower (Fig 3). Nonetheless, the best acoustic emission signals tend to be seen more anteriorly, but with proper adjustment of the color box, focus (Fig 5), and output power (ie, use of a mechanical index > 0.5), the effects can be seen throughout the liver, at least at the relatively shallow (<5-cm) depths examined in this study. Finally, it should be remembered that SHU 563A circulates in the blood vessels for 5–8 minutes initially. Therefore, when using the acoustic emission characteristics of SHU 563A, it is important to wait at least 15 minutes after administration to ensure that there is sufficient uptake of the agent in the RES.

By using the distinctive characteristics of acoustic emission signals, we demonstrated a marked and statistically significant improvement in the detectability of hepatic tumors after contrast agent administration (Tables 1, 2). CDI enabled a clear delineation of tumors as colorfree regions owing to the lack of contrast agent uptake (Figs 5–8). As might be expected, it was easier to delineate larger lesions; however, masses as small as 4 x 3 mm were identified (Fig 8). There were no false-positive results, although the size of one tumor was overestimated at baseline US. Detection rates increased from 33% (four of 12 lesions) to 100% (P = .0078) and from 45% (nine of 20 lesions) to 85% (17 of 20 lesions) (P = .0215) for VX-2 tumors in rabbits and for hepatocellular carcinomas in woodchucks, respectively.

It was easier to detect VX-2 tumors, because it is known that at least one mass will be present. Consequently, the sensitivity calculated for detecting rabbits with tumors—57% [four of seven rabbits] before and 100% after contrast agent administration—can be considered as a guideline only because of the limited number of animals and the investigator bias. In these studies, we investigated lesion detection rather than overall diagnostic accuracy; for example, there were no tumorfree animals in this part of the study. Woodchuck hepatomas were more challenging to detect because no prior information on the number of tumors existed, and there were up to 11 lesions in each animal. This increased difficulty was confirmed by the results (Table 2). There was no relationship between tumor diameter and tumor detectability in this study.

A tissue-specific US contrast agent, SHU 563A, has been evaluated in vitro and in vivo. CDI with this agent demonstrates a random mosaic color pattern—that is, induced acoustic emission—even when no flow is present. The uptake of SHU 563A in the RES in vivo and the characteristic appearance of acoustic emission signals provide a distinctive method of visualizing normal hepatic and splenic parenchyma. Acoustic emission signals also improve the differentiation of normal liver tissue from abnormal liver tissue and enhance the detectability of hepatic tumors. This type of tissue-specific US contrast agent shows great promise in the evaluation of a variety of abnormalities of the liver and spleen.


Practical Application.
There are limitations to the generation of acoustic emission signals with SHU 563A because the sound energy destroys the bubbles within the cells. Thus, after the initial sonification of an area, resonification will not result in the same clearly defined mosaic pattern. This problem can be overcome, however, by using additional injections of the contrast agent, which will result in a reaccumulation of the contrast agent in the normal tissues (as well as a prolonged study). It is likely that achieving sufficient acoustic pressure to rupture the contrast agent bubbles in human livers, which are located more deeply in the body, will be more difficult. However, the use of lower-frequency transducers may partially alleviate this problem. In humans, it is possible that lesions as small as 4 x 3 mm will not be detectable. However, even with contrast-enhanced computed tomography (CT) and magnetic resonance (MR) imaging, it is believed that tumors smaller than 5 mm cannot be detected consistently (29–32).

Combining several of the different characteristics of SHU 563A during the first and second phases of enhancement could possibly lead to the ability to differentiate a large variety of tumors. For instance, it is known that primary hepatocellular carcinomas are fed primarily by the hepatic artery. Thus, in the first enhancement phase, the flow of the contrast agent should be seen in the hepatic arteries first and then in the portal and hepatic veins, which is usually not the case with metastatic tumors (33,34). Because focal nodular hyperplasia involves normal liver cells, it would show the same acoustic emission characteristics as those of normal hepatic tissue while it manifests as a mass.

It is also conceivable that SHU 563A could enable the identification of hemangiomas. It is well known that there is very slow blood flow within these masses (34–36). Thus, it is difficult to identify flow in hemangiomas by using Doppler US in almost all cases. However, over time, SHU 563A would accumulate in the slow-flowing vessels in these masses. Thus, by using similar timing sequences as those that have been shown to be effective with dynamic CT, MR imaging, and nuclear scanning (29–34,36–38), it should be possible to demonstrate SHU 563A in the hemangioma, relying on the same acoustic emission effect as that when the agent is trapped within the RES. The hemangioma would not be expected to demonstrate substantial flow initially, but after a delay, it would be expected to show acoustic emission signals within the mass, assuming that the initial scanning did not destroy contrast to a degree that limits visualization of the accumulation phase. This enhancement pattern would be different from the pattern seen with hepatocellular carcinoma or metastatic tumor, in which delayed scanning should demonstrate an absence of the mosaic color pattern.


   Footnotes
 
Supported in part by a grant from Schering, Berlin, Germany

Address reprint requests to F.F.

From the 1995 RSNA scientific assembly.

Abbreviations: CDI = color Doppler imaging RES = reticuloendothelial system

Author contributions: Guarantor of integrity of entire study, F.F.; study concepts and design, F.F., B.B.G.; definition of intellectual content, F.F., B.B.G.; literature research, F.F., J.B.L.; clinical studies, F.F., B.B.G., J.B.L., D.A.M., N.M.R.; experimental studies, F.F., W.T.S.; data acquisition, F.F., B.B.G., J.B.L., D.A.M., N.M.R., W.T.S.; data analysis, F.F., B.B.G., D.A.M., J.B.L.; statistical analysis, F.F.; manuscript preparation, editing, and review, F.F., B.B.G., J.B.L., D.A.M., N.M.R., W.T.S.

Received September 25, 1997; revision requested December 15, 1997; revision received July 2, 1997; accepted August 24, 1997.
   References
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Ophir J, Parker KJ. Contrast agents in diagnostic ultrasound. Ultrasound Med Biol 1989; 15:319-333.[Medline]
  2. Goldberg BB, Liu JB, Forsberg F. Ultrasound contrast agents: a review. Ultrasound Med Biol 1994; 20:319-333.[Medline]
  3. Needleman L, Forsberg F. Contrast agents in ultrasound. Ultrasound Q 1996; 13:121-138.
  4. Crouse LJ, Cheirif J, Hanley DE, et al. Opacification and border delineation improvement in patients with suboptimal endocardial border definition in routine echocardiography: results of the phase III Albunex (multicenter trial). J Am Coll Cardiol 1993; 22:1494-1500.[Abstract]
  5. Goldberg BB, Liu JB, Burns PN, Merton DA, Forsberg F. Galactose-based intravenous sonographic contrast agent: experimental studies. J Ultrasound Med 1993; 12:463-470.[Abstract]
  6. Kedar RP, Cosgrove D, McCready VR, Bamber JC, Carter ER. Microbubble contrast agent for color Doppler US: effect on breast masses. Radiology 1996; 198:679-686.[Abstract/Free Full Text]
  7. Goldberg BB. Ultrasound contrast agents London, England: Dunitz, 1997.
  8. Forsberg F, Liu JB, Merton DA, Rawool NM, Goldberg BB. Parenchymal enhancement and tumor visualization using a new sonographic contrast agent. J Ultrasound Med 1995; 14:949-957.[Abstract]
  9. Brown JM, Taylor KJW, Alderman JL, Quedens-Case C, Greener Y. Contrast-enhanced ultrasonographic visualization of gonadal torsion. J Ultrasound Med 1997; 16:309-316.[Abstract]
  10. Metzger-Rose C, Krupinski EA, Wright WH, et al. Ultrasonographic detection of testicular ischemia in a canine model using phospholipid coated microbubbles (MRX-115). J Ultrasound Med 1997; 16:317-324.[Abstract]
  11. Mattrey RF. Perfluorooctylbromide: a new contrast agent for CT, sonography, and MR imaging. AJR 1989; 152:247-252.[Free Full Text]
  12. Behan M, OConnell D, Mattrey RF, Carney DN. Perfluorooctylbromide as a contrast agent for CT and sonography: preliminary results. AJR 1993; 160:399-405.[Abstract/Free Full Text]
  13. Fritzsch T, Heldmann D, Reinhardt M. The potential of a novel ultrasound contrast medium. In: Goldberg BB, eds. Ultrasound contrast agents. London, England: Dunitz, 1997; 169-176.
  14. Goldberg BB, Forsberg F, Fritzsch T, Liu JB, Merton DA, Rawool NM. Evaluation of a US contrast agent employing induced acoustic emission (abstr). Radiology 1995; 197(P):402.[Free Full Text]
  15. Thorstensen O, Isberg B, Svahn V, Jorulf H, Venizelos N, Jaremko G. Experimental tissue transplantation using a biopsy instrument and radiologic methods. Invest Radiol 1994; 29:469-471.[Medline]
  16. Snyder RL, Tyler G, Summers J. Chronic hepatitis and hepatocellular carcinoma associated with woodchuck hepatitis virus. Am J Pathol 1982; 107:422-425.[Medline]
  17. Hedrick WR, Hykes DL. An overview of thermal and mechanical acoustic output indices. JDMS 1993; 9:228-235.
  18. Rosner B. Fundamentals of Biostatistics 3rd ed. Boston, Mass: PWS-Kent, 1990.
  19. Uhlendorf V, Hoffmann C. Nonlinear acoustical response of coated microbubbles in diagnostic ultrasound. Proc IEEE Ultrasound Symp 1994; :1559-1562.
  20. Porter TR, Xie F. Transient myocardial contrast after initial exposure to diagnostic ultrasound pressures with minute doses of intravenously injected microbubbles. Circulation 1995; 92:2391-2395.[Abstract/Free Full Text]
  21. Moriyasu F, Kono Y, Nada T, Matsumura T, Suginoshita Y, Kobayashi K. Flash echo (passive cavitation) imaging of the liver by using US contrast agents and intermittent scanning sequence (abstr). Radiology 1996; 201(P):196.
  22. Goldberg BB, Merton DA, Deane CR. An atlas of ultrasound color flow imaging London, England: Dunitz, 1997.
  23. Miyatake K, Yamagishi M, Tanaka N, et al. New method for evaluating left ventricular wall motion by color-coded tissue Doppler imaging: in vitro and in vivo studies. J Am Coll Cardiol 1995; 25:717-724.[Abstract]
  24. Forsberg F, Liu JB, Burns PN, et al. Artifacts in ultrasonic contrast agent studies. J Ultrasound Med 1994; 13:357-365.[Abstract]
  25. Lanza GM, Wallace KD, Scott MJ, et al. A novel site-targeted ultrasonic contrast agent with broad biomedical application. Circulation 1997; 95:3334-3340.
  26. Unger EC, McCreery TP, Shen DK, Wu GL, Sweitzer RH, Yellowhair D. Thrombus specific Aerosomes: a novel ultrasound contrast agent (abstr). J Ultrasound Med 1997; 16:S36.
  27. Forsberg F, Goldberg BB, Liu JB, Merton DA, Fujimoto M, Johnson DK. Detection of hepatic VX-2 tumors in rabbits with ultrasound contrast and induced acoustic emission (abstr). Radiology 1997; 205(P):279.
  28. Blomley MJ, Albrecht T, Cosgrove DO, et al. Stimulated acoustic emission with the echo-enhancing agent Levovist: a reproducible effect with potential clinical utility (abstr). Radiology 1997; 205(P):278-279.[Abstract/Free Full Text]
  29. Mitsuzaki K, Yamashita Y, Ogata I, Nishiharu T, Urata J, Takahashi M. Multiple-phase helical CT of the liver for detecting small hepatomas in patients with liver cirrhosis: contrast-injection protocol and optimal timing. AJR 1996; 167:753-757.[Abstract/Free Full Text]
  30. Reimer P, Rummeny EJ, Daldrup HE, et al. Enhancement characteristics of liver metastases, hepatocellular carcinoma, and hemangiomas with Gd-EOB-DTPA: preliminary results with dynamic MR imaging. Eur Radiol 1997; 7:275-280.[Medline]
  31. Murakami T, Baron RL, Peterson MS, et al. Hepatocellular carcinoma: MR imaging with mangafodipir trisodium (Mn-DPDP). Radiology 1996; 200:69-77.[Abstract/Free Full Text]
  32. Itai Y, Ohtomo K, Furui S, Yamauchi T, Minami M, Yashiro N. Noninvasive diagnosis of small carvernous hemangioma of the liver: advantage of MRI. AJR 1985; 145:1195-1199.[Abstract/Free Full Text]
  33. Harisinghani MG, Saini S, Weissleder R, et al. Differentiation of liver hemangiomas from metastases and hepatocellular carcinoma liver at MR imaging enhanced with blood-pool contrast agent code-7227. Radiology 1997; 202:687-691.[Abstract/Free Full Text]
  34. Itai Y, Matsui O. Blood flow and liver imaging. Radiology 1997; 202:306-314.[Free Full Text]
  35. Urhahn R, Kilbinger M, Drobnitzky M, Mans-Peine G, Neuerburg J, Gunther RW. Dynamic Gd-enhanced MR imaging of hepatic hemangioma: is high temporal resolution requisite for characterization?. Magn Reson Imaging 1996; 14:31-41.[Medline]
  36. Brooks M. The liver. In: Goldberg BB, Pettersson H, eds. Nicer Yearbook 1996: Ultrasonography. Oslo, Norway: NICER Foundation, 1996; 55-81.
  37. Liou J, Lee K, Borello JA, Brown JJ. Differentiation of hepatomas from nonhepatomatus masses: use of Mn DPDP-enhanced MR images. Magn Reson Imaging 1994; 12:71-79.[Medline]
  38. Berger JF, Laissy JP, Limot O, et al. Differentiation between multiple liver hemangiomas and liver metastases of gastrinomas: value of enhanced MRI. J Comput Assist Tomogr 1996; 20:349-355.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Roentgenol.Home page
S. H. Kim, J. M. Lee, J. Y. Lee, J. K. Han, and B. I. Choi
Contrast-Enhanced Sonography of Intrapancreatic Accessory Spleen in Six Patients
Am. J. Roentgenol., February 1, 2007; 188(2): 422 - 428.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Radiol.Home page
V R Stewart and P S Sidhu
New directions in ultrasound: microbubble contrast.
Br. J. Radiol., March 1, 2006; 79(939): 188 - 194.
[Full Text] [PDF]

Home page
 Am. J. Roentgenol.Home page
C. Gorg and T. Bert
Second-generation sonographic contrast agent for differential diagnosis of perisplenic lesions.
Am. J. Roentgenol., March 1, 2006; 186(3): 621 - 626.
[Abstract] [Full Text] [PDF]

Home page
 J Ultrasound MedHome page
C. Gorg, T. Bert, and R. Kring
Contrast-Enhanced Sonography of the Lung for Differential Diagnosis of Atelectasis
J. Ultrasound Med., January 1, 2006; 25(1): 35 - 39.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
C. Gorg, T. Bert, and K. Gorg
Contrast-Enhanced Sonography for Differential Diagnosis of Pleurisy and Focal Pleural Lesions of Unknown Cause
Chest, December 1, 2005; 128(6): 3894 - 3899.
[Abstract] [Full Text] [PDF]

Home page
 J Ultrasound MedHome page
J. S. Abramowicz
Ultrasonographic Contrast Media: Has the Time Come in Obstetrics and Gynecology?
J. Ultrasound Med., April 1, 2005; 24(4): 517 - 531.
[Abstract] [Full Text] [PDF]

Home page
 RadiologyHome page
A. K. P. Lim, N. Patel, R. J. Eckersley, S. D. Taylor-Robinson, D. O. Cosgrove, and M. J. K. Blomley
Evidence for Spleen-specific Uptake of a Microbubble Contrast Agent: A Quantitative Study in Healthy Volunteers
Radiology, June 1, 2004; 231(3): 785 - 788.
[Abstract] [Full Text] [PDF]

Home page
 J Ultrasound MedHome page
A. von Herbay, C. Vogt, and D. Haussinger
Differentiation Between Benign and Malignant Hepatic Lesions: Utility of Color Stimulated Acoustic Emission With the Microbubble Contrast Agent Levovist
J. Ultrasound Med., February 1, 2004; 23(2): 207 - 215.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Radiol.Home page
H-D Liang and M J K Blomley
The role of ultrasound in molecular imaging
Br. J. Radiol., December 1, 2003; 76(suppl_2): S140 - S150.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Roentgenol.Home page
A. von Herbay, C. Vogt, and D. Haussinger
Late-Phase Pulse-Inversion Sonography Using the Contrast Agent Levovist: Differentiation Between Benign and Malignant Focal Lesions of the Liver
Am. J. Roentgenol., November 1, 2002; 179(5): 1273 - 1279.
[Abstract] [Full Text] [PDF]

Home page
 ImagingHome page
A S Shaw, S M Ryan, R C Beese, S Norris, M Bowles, M Rela, and P S Sidhu
Liver transplantation
Imaging, August 1, 2002; 14(4): 314 - 328.
[Abstract] [Full Text] [PDF]

Home page
 J Ultrasound MedHome page
J.-B. Liu, D. A. Merton, B. B. Goldberg, N. M. Rawool, W. T. Shi, and F. Forsberg
Contrast-Enhanced Two- and Three-dimensional Sonography for Evaluation of Intra-abdominal Hemorrhage
J. Ultrasound Med., February 1, 2002; 21(2): 161 - 169.
[Abstract] [Full Text] [PDF]

Home page
 RadiologyHome page
M. J. K. Blomley, P. S. Sidhu, D. O. Cosgrove, T. Albrecht, C. J. Harvey, R. A. Heckemann, J. Butler-Barnes, R. J. Eckersley, and R. Basilico
Do Different Types of Liver Lesions Differ in Their Uptake of the Microbubble Contrast Agent SH U 508A in the Late Liver Phase? Early Experience
Radiology, September 1, 2001; 220(3): 661 - 667.
[Abstract] [Full Text] [PDF]

Home page
 BMJHome page
M. J K Blomley, J. C Cooke, E. C Unger, M. J Monaghan, and D. O Cosgrove
Science, medicine, and the future: Microbubble contrast agents: a new era in ultrasound
BMJ, May 19, 2001; 322(7296): 1222 - 1225.