Brief Report

Contrast-enhanced ultrasound for blunt hepatic trauma: an animal experiment^?

Li Yekuo MD ^a,?, Wang Shasha PhD ^a, Zhu Xiansheng MM ^a, Chen Qi MM ^a,

Luo Guoxin MM ^a, Huo Feng PhD ^b

^aDepartment of Ultrasound, Liuhuaqiao Hospital, Guangzhou 510010, China

^bDepartment of Hepatobiliary Surgery, Liuhuaqiao Hospital, Guangzhou 510010, China

Received 17 June 2009; revised 29 July 2009; accepted 30 July 2009

Abstract

Objective: The aim of this study was to study the value of contrast-enhanced ultrasound (CEUS) in the diagnosis of Active hemorrhage and intraparenchymal lesions in blunt hepatic trauma versus conventional ultrasound (US).

Methods: Twenty heparinized and anesthetized domestic pigs have been created to animal models with blunt hepatic trauma by a special impacting device. Conventional US and CEUS were performed to determine if hepatic traumas were present.

Results: Active hemorrhage, the presence of intraparenchymal lesions, and sonographic pattern were evaluated for conventional US and CEUS, as compared with laparotomy and pathologic findings. Contrast-enhanced US detected active hemorrhage from the injured livers in all cases, but conventional US did not find that in any case. The sensitivity of CEUS and conventional US in diagnosing intraparenchymal lesions of blunt hepatic trauma were 100% and 60%, respectively.

Conclusions: Contrast-enhanced US is more sensitive than conventional US in determining the active hemorrhage and intraparenchymal lesions in blunt hepatic trauma.

Crown Copyright (C) 2010 Published by

Introduction

Trauma is the leading cause of death in a population younger than 45 years [1]. Given the morbidity and mortality of trauma, prompt assessment and diagnosis are crucial. Computed tomography (CT) has been shown to be valuable for evaluating liver injury in patients with blunt hepatic trauma [2,3]. It was advocated as the routine method of choice in stable and cooperative patients with blunt hepatic

^? This work was supported by the Natural Science Foundation of Guangdong Province of China (06019712).

* Corresponding author. Tel.: +86 20 36653449.

E-mail address: yekuoli@hotmail.com (L. Yekuo).

trauma [4]. However, CT is not convenient to be used at the patients’ bedsides and the accident sites because of its cumbersome imaging and high exposure to ionizing radiation [5-9].

Conventional ultrasound (US) is a good modality in the trauma setting for its portable, rapid, noninvasive, and practical nature. At present, conventional US is widely used as a screening tool for free abdominal and pericardial fluid in cases of abdominal trauma (focused assessment sonography in trauma). However, conventional US cannot reliably determine the site of the hemorrhage from the injured liver, and it is not sensitive for the detection of intraparenchymal lesions of the abdominal solid organs [10]. Conventional US is not adequately useful to the surgeon when treatment on blunt hepatic trauma had to be planned.

0735-6757/$ – see front matter. Crown Copyright (C) 2010 Published by doi:10.1016/j.ajem.2009.07.028

histologic examination“>Contrast-enhanced ultrasound (CEUS) is based on the ability of microbubbles containing gases to produce real- time, contrast-related, gray-scale images. The development of US Contrast agents in combination with the development of US equipment has overcome the limitations of conven- tional US and enable the display of liver parenchymal microvaculature [11-14]. SonoVue (Bracco, Italy) we used in our study is a perfluoro gas filled with microbubble, which has been reported to enhance liver parenchymal traumas in the emergency setting [15-17]. The character- istics of hemorrhage of blunt hepatic trauma under CEUS are rarely reported.

The purpose of this animal study was to compare the performances of CEUS to conventional US regarding the detection of the active hemorrhage and intraparenchymal lesions in blunt hepatic trauma.

Methods

Animal models

This study was preapproved by the research animal care and use committee of the Liuhuaqiao Hospital. This study was performed in 20 healthy Guangzhou domestic pigs weighing 45 to 55 kg. The animals were initially anesthetized with ketamine hydrochloride (50 mg/kg) by Intramuscular injection. Additional injections with 2% pentobarbital (0.25 mg/kg) were conducted to maintain effective anesthesia for at least 1 hour. They were intubated and ventilated with oxygen and 1% to 2% isoflurance, with the concentration being varied to maintain normal vital signs. The pigs were heparinized before injury, with an Intravenous bolus of 200 U/kg to create persistent active hemorrhage after the impact during the whole experiment.

Blunt hepatic trauma was produced following a tech- nique reported by Cohn et al [18]. A nail-driving gun (SDQ307; Nanshan, Sichuan, China) was modified to impact the pigs to produce the animal models with blunt Liver trauma. The muzzle of the gun was placed and fired against 2-cm-diameter 2-cm-thick iron cylinder (weight, 300 g). The gun was loaded with the S5 (5.6 x 16.0 mm) charge. The compression depth was set at 4 cm. The iron cylinder was positioned and oriented in the preferable direction on the abdomen 10 cm under the xiphoid in the upper quadrant over the region of the liver and then the gun was fired. After the impact, the pigs remained monitored in the operation room.

Each pig was observed under general anesthesia for 10 minutes after the examination of conventional US and CEUS. For the killing of the pigs, a dose of 1.0 g thiopental was administered under general anesthesia. After the death of the pigs, the liver was harvested and the injured liver intraparenchymal lesions were measured and sent for micropathologic examination.

US examination

Conventional US examination was performed with a Sequoia 512 scanner (Siemens Ultrasound, USA) equipped with contrast pulse sequencing software, which permits real- time depiction of lesion blood perfusion under low mechanical index. 4C1-S transducer, 2-4 MHz, was used. The entire liver was scanned using conventional US 10 minutes after the impact.

Contrast-enhanced US was performed immediately after conventional US. We used US contrast agent SonoVue (Bracco, Milan, Italy), which was supplied as a lyophilized powder and reconstituted with 5 mL of saline to form a homogeneous microbubble suspension. Contrast pulse sequencing was activated after the administration of 1.2 mL of microbubble suspension as a bolus through a 20- gauge cannula placed in the femoral vein. Every injection was followed by a 5-mL saline flush. The range of the mechanical index was 0.18 to 0.21. The complete CEUS examination lasted no more than 5 minutes. The results of conventional US and CEUS examination were stored in a hard disk and transferred to a personal computer for subsequent offline analysis.

Pathologic and histologic examination

The damaged region paralleling the impact direction was measured with calipers in the fresh liver ex vivo before preservation. Next, the specimens were preserved in 10% formalin solution for microscopic analysis. Represen- tative tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin and evaluated under light microscope.

Image analysis

Three radiologists who blinded to the trauma results analyzed the CEUS and conventional US results in a Tomtec imaging station (Tomtec Imaging Systems Inc, Bolder, CO). After the injection of contrast agents, the intraparenchyma was considered injured when its enhancing characteristic was different to the surrounding parenchyma, which was classified as hyperenhancing, isoenhancing, or hypoenhan- cing. The time to enhancement and the type of enhancement of each lesion were recorded. Due to the specific Blood supply to the liver, 3 contrast phases can be differentiated: the arterial pHase, which starts 10 to 30 seconds after intravenous injection; the portal venous pHase, which extends from 30 to 35 seconds postinjection to 120 seconds; and the late phase begins, which ends with the disappearance of the bubbles (approximately 5 minutes postinjection). Active hemorrhage was defined as US evidence of Contrast material collection with echogenicity similar to that of the adjacent vessel and greater than that of the surrounding parenchyma [19-21]. Conventional US findings were

considered positive when the lesion’s echo was different compared with surrounding parenchyma, which was defined as a hyperechoic or hypoechoic. The damaged intrapar- enchymal region paralleling the impact direction was measured under the CEUS and conventional US review.

Statistical analysis

Means, SDs, and ranges were used as descriptive statistics. To calculate the sensitivity, pathologic results were considered the criterion standard. The Fisher exact test was used to compare the diagnostic capability of conventional US with that of CEUS. Statistical analyses were performed on a personal computer with the SPSS statistical package (SPSS 13.0 for Windows, SPSS,

Chicago, IL). Two-tailed P values less than .05 were considered significant.

Results

Conventional US findings

Conventional US detected 12 intraparenchymal lesions ranging from 2.0 to 4.5 cm (mean +- SD, 3.5 +- 0.8 cm), of whom 8 were hyperechoic and 4 were hypoechoic; the sensitivity was 60% (12/20). The borders of the intraparench- ymal lesions were poorly defined by conventional US. Active bleeding was not observed in any case. The fluid around the injured liver was clearly found in each pig (Fig. 1A).

Fig. 1 Conventional US and CEUS on blunt hepatic trauma. A, Intraparenchymal lesion and free fluid were demonstrated with hypoechoic, but active bleeding was not detected on conventional US. B, Arterial phase: extraparenchymal hemorrhage was shown with slightly hyperenhancing, and intraparenchymal lesion was found with hypoenhancing. C, Portal phase: a jet-like extraparenchymal hemorrhage and a pool-like intraparenchymal bleeding were detected. D, Late phase: the margins of the intraparenchymal lesion were clearly demonstrated.

CEUS findings

Contrast-enhanced US found that the intraparenchymal lesions ranged from 5.5 to 8.0 cm (mean +- SD, 7.0 +- 1.1 cm) in all of the 20 pigs; the sensitivity was 100% (20/20). The margins of these intraparenchymal lesions starting from arterial to late phase were hypoenhancing and were clearly shown (Fig. 1B-D).

Contrast-enhanced US detected active hemorrhages in all cases, which appeared as contrast material collection, with echogenicity greater than that of the surrounding parenchy- ma. After the injection of US contrast agent, a jet-like extraparenchymal hemorrhage and a pool-like intraparench- ymal bleeding were found in each pig (Fig. 1C).

Laparotomy and pathologic findings

Laparotomy and gross pathologic examinations con- firmed that the 20 pigs suffered from blunt hepatic trauma. All of the liver lesions were located in the left hepatic lobe ranging from 6.0 to 8.5 cm (mean +- SD, 7.5 +- 1.0 cm). Capsular tears, active hemorrhages, intraparenchymal lesions, and free fluid were found in all cases (Fig. 2A, B). histopathologic examination observed a few shattered hepatic plate and many neutrophils and degenerated hepatocytes in the injured lesions.

Using the Fisher exact test, we found that in comparison with conventional US, CEUS showed significantly more sensitivity in detecting active hemor- rhage and intraparenchymal lesion in blunt hepatic trauma (P b .05).

Discussion

It is crucial to rapidly and accurately diagnose the hemorrhage and intraparenchymal lesions in patients with blunt hepatic trauma because these warranted an immediate treatment. Conventional US in the evaluation of abdominal trauma evolved greatly over the past 40 years [22-25]. It is highly sensitive to the presence of free peritoneal fluid, but it is less sensitive for the detection of active hemorrhage and parenchymal lesions of the abdominal solid organs [26-28]. Overall, contrast-enhanced CT has been considered superior over conventional US in diagnosing blunt abdominal trauma. Many comparative studies have focused on the availability of Contrast enhancement of the circulation of the injured liver for CT and the lack of corresponding contrast enhancement for US. With the development of US contrast agents and specific imaging modes, they offer the potential of solving some of the limitations of imaging trauma with conventional US.

Clinical US contrast agents must be able to cross the lung bed to produce systemic enhancement and render them sufficient stability as they persist for several minutes after intravenous injection. Both the gas they contain (usually air or a perfluoro gas) and the stabilizing shell (denatured albumin, surfactants, or phospholipids) are critical to their effectiveness. Albunex is the first widely used US contrast agent. It has an albumen shell and contains air, which is mainly used in cardiology [29]. Levovist (Schering, Berlin) is the first agent for general use. It is made of galactose microcrystals, of which the surfaces provide nidation sites on which air bubbles form when they are suspended in water [30]. SonoVue is one of the most widely used US contrast

Fig. 2 Gross pathologic pictures of blunt hepatic trauma. A, Several parenchymal tears with active bleeding were found on laparotomy. B, Intraparenchymal lesion was shown on gross pathologic examination.

agents in current clinical practice. It is one of the family members of perfluoro gas-containing agent that use phospholipids as the membrane. SonoVue has been shown to be safe, and the reporting rate of serious adverse events was 0.0086% [31]. It has been proved useful for diagnosing benign and malignant focal liver lesions [32,33]. SonoVue offers the potential of solving some of the limitations of imaging liver trauma with conventional US.

In our study, 100% (20/20) active hemorrhages have been detected from the blunt injured livers after the administration of CEUS. However, conventional US did not find ongoing hemorrhage in any case. SonoVue we used in this experiment does not disrupt on exposure to low-energy US. This property enables the circulating of microbubbles with blood in the body so that real-time monitoring of macrovascular and microvascular dynamics can be performed in the liver in the same phase of perfusion. Therefore, the extravasated blood from the damaged liver can be clearly observed in real time after the injection of SonoVue. The ability of CEUS to depict extravasation of US contrast agents within the damaged lesions makes it possible to find the active bleeding sites, which is very helpful to provide the trauma surgeons with important information for next treatment.

In this experiment, the intraparenchymal lesions in the injured livers can be clearly identified with CEUS. The sensitivity of CEUS was significantly higher than those of conventional US, with good agreement with pathologic findings. Ultrasound contrast agents are blood pool agents that are used to identify subtle vascular alterations. After the impact, the blood flow in the intraparenchymal lesions was severely reduced as compared with surrounding normal hepatic parenchyma. The blood cannot adequately perfuse the damaged lesions; thus, the intraparenchymal lesions seemed hypoenhancing after the injection of the US agents from the artery phase to late phase. The borders of the intraparenchyma lesions at CEUS were clearer than that at conventional US, and the sizes of the intraparenchymal lesions of the CEUS findings were bigger than that of the conventional US.

In conclusion, our study shows that CEUS is more sensitive than conventional US in the diagnosis of the active hemorrhage and intraparenchymal lesions in blunt hepatic trauma. Further prospective series are required to confirm our findings.

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