Corresponding author: Mitsuyoshi Takiguchi, Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18 W9 Sapporo, Hokkaido 060 0818, Japan; e-mail: email@example.com.
Background: Contrast-enhanced ultrasonography with perflubutane microbubbles improves the diagnostic accuracy to differentiate benign and malignant focal liver lesions in dogs.
Hypothesis: Perflubutane microbubbles-enhanced ultrasonography is useful for differentiation of benign from malignant focal splenic lesions in dogs.
Animals: Twenty-nine clinical dogs with single or multiple focal splenic lesions detected by conventional ultrasonography.
Methods: Prospective clinical observational study. Perflubutane microbubbles-enhanced ultrasonography was performed in 29 dogs with focal splenic lesions. Qualitative assessment of the enhancement pattern was performed in the early vascular, late vascular, and parenchymal phases.
Results: In the early vascular phase, a hypoechoic pattern was significantly associated with malignancy (P= .02) with sensitivity of 38% (95% confidence interval [CI], 25–38%) and specificity of 100% (95% CI, 84–100%). In the late vascular phase, a hypoechoic pattern was significantly associated with malignancy (P= .001) with sensitivity of 81% (95% CI, 66–90%) and specificity of 85% (95% CI, 65–95%). There was no significant difference between malignant and benign lesions during the parenchymal phase.
Conclusions and Clinical Importance: Hypoechoic splenic nodules in the early and late vascular phases with perflubutane microbubbles-enhanced ultrasonography are strongly suggestive of malignancy in dogs.
Ultrasonography is a routine diagnostic procedure for the detection of focal splenic lesions. However, differentiation between benign and malignant lesions is difficult in many cases using conventional ultrasonography, and clear discrimination criteria are lacking.1 Therefore, histologic or cytologic examination is necessary to confirm the diagnosis of splenic lesions.
Contrast-enhanced ultrasonography is a new diagnostic imaging procedure to investigate tissue vascularity and perfusion dynamics. A major advance of contrast-enhanced ultrasonography is the development of contrast-specific software allowing pulse subtraction imaging, and the development of second-generation contrast agents, which allow low-mechanical index (MI) ultrasonography. First-generation contrast agents have contrast effect only at high MI leading to microbubble destruction.2 Therefore, continuous or high-frame rate observation cannot be performed with first-generation contrast agents. On the other hand, second-generation contrast agents are of diagnostic value even with a very low MI because of their physical behavior during insonation. Second-generation contrast agent perflubutane microbubblesa produce stable contrast effect at low MI without destruction and allow continuous real-time imaging.3 Previous studies showed that contrast-enhanced ultrasonography with second-generation contrast agents could evaluate the tumor perfusion dynamics and improve the differentiation between benign and malignant focal splenic lesions in humans4 and dogs.5–7
In addition to vascular imaging, the second-generation contrast agent perflubutane microbubbles is phagocytized by Kupffer cells,8,9 allowing for long-lasting parenchymal contrast enhancement of the liver.10 Parenchymal imaging improves the diagnostic accuracy to differentiate benign and malignant focal liver lesions in humans3,11,12 and dogs,13,14 given that malignant tumors have little or no reticuloendothelial system and appear as hypoechoic defects.
Perflubutane microbubbles allow for parenchymal imaging of the normal spleen in dogs.15 The purpose of this study was to determine whether analysis of the enhancement pattern after injection of perflubutane microbubbles would allow one to differentiate benign from malignant focal splenic lesions.
Materials and Methods
Twenty-nine focal splenic lesions in 29 dogs were examined. The study population was recruited from dogs with single or multiple focal splenic lesions detected by conventional ultrasonography in the Veterinary Teaching Hospital of the Graduate School of Veterinary Medicine, Hokkaido University. The final diagnosis was confirmed by histology or cytology.
Contrast-enhanced ultrasonography was performed with a ultrasound machineb with a 5–11 MHz broadband linear probec or a 3.75 MHz convex probe.d A single focal zone was placed at the deepest part of the lesion. The MI was set at 0.1–0.2 MI to minimize microbubble destruction. The gain was set so that few signals from the underlying splenic parenchyma were present. The cranial abdomen was shaved and the dogs were restrained in dorsal recumbency. Scan planes were chosen to show both a splenic lesion and normal parenchyma in 1 image. Perflubutane microbubbles (0.12 μL microbubbles/kg) was injected via an intravenous catheter in the cephalic vein. Catheters were flushed with saline (0.9% NaCl) solution immediately after the injection. Real-time imaging was performed from preinjection to 1 minute after injection of perflubutane microbubbles for the vascular phases. Images for parenchymal phase were obtained 7–10 minutes after injection according to our previous study.15 All images were recorded on a hard disk for off-line analysis.
Qualitative assessment of the vessel appearance was performed immediately after injection of perflubutane microbubbles. Vessel appearance in lesion was divided into 3 groups in comparison with the vessel in surrounding normal parenchyma: (1) similar, (2) different, and (3) invisible.
Qualitative assessment of the enhancement pattern was performed in the early vascular phase (5–10 seconds after injection), late vascular phase (25–30 seconds after injection), and parenchymal phase (7–10 minutes after injection). The timing of all 3 phases was defined based on the results of our previous study.15 The contrast enhancement in the surrounding parenchyma was used as an in vivo reference. Enhancement pattern were defined subjectively based on echogenicity of lesion parenchyma in comparison with the surrounding normal parenchyma: (1) hypoechoic, (2) isoechoic, and (3) heteroechoic.
Diagnosis was confirmed in all 29 dogs, by histology in 24 dogs and ultrasound-guided aspiration cytology in 5 dogs. It was ensured by size and location that the lesions imaged were the ones sampled.
The statistical significance of differences between benign and malignant lesions was calculated with the G-test and 2-tailed Fisher's exact test. In addition, the sensitivity and specificity were calculated with 95% confidence intervals (CI). P < .05 was considered significant. Statistical analysis was performed with a standard computer software program.e
Twenty-nine dogs were included in this study. Of the 29 dogs, 13 dogs had benign nodules and 16 dogs had malignant tumors (Table 1).
Table 1. Clinical data of 29 dogs with splenic lesions.
Median Age (years)
Median Diameter (mm)
Male (14), Female (15)
Single (18), Multiple (11)
Histology (24), Cytology (5)
Male (9), Female (4)
Single (8), Multiple (5)
Histology (11), Cytology (2)
Male (5), Female (3)
Single (5), Multiple (3)
Histology (6), Cytology (2)
Male (1), Female (1)
Male (5), Female (11)
Single (10), Multiple (6)
Histology (13), Cytology (3)
Male (3), Female (5)
Male (1), Female (2)
Male (1), Female (1)
The vessel appearance was not significantly different between malignant and benign lesions. Similar pattern was found in 9 of the 16 malignant lesions (Fig 4B) and 10 of the 13 benign lesions (Fig 1B). Different pattern was found in 4 malignant lesions. In the other 3 malignant and 3 benign lesions, vessel was invisible (Fig 2B). Among 4 dogs with different pattern, there were 3 hemangiosarcoma and 1 histiocytic sarcoma. All 3 hemangiosarcoma had aberrant wide or tortuous vessels in nodule (Fig 3B and C). One histiocytic sarcoma had tortuous vessel. Data for the vessel appearance are given in Table 2.
Table 2. Contrast enhancement pattern in 29 dogs.
Significantly different between benign and malignant lesions : aP < .001 ; bP= .02. Statistical differences were calculated with G-test. Significantly different between benign and malignant lesions : cP= .001 ; dP= .02. Statistical differences were calculated with 2-tailed Fisher's exact-test.
The enhancement patterns during the early and late vascular phases were significantly different between malignant and benign lesion (P= .02 and P < .001). There was no significant difference during the parenchymal phase. Data for the contrast enhancement pattern after perflubutane microbubbles injection are given in Table 2.
In the early vascular phase, a hypoechoic pattern was found in 6 of the 16 malignant lesions and in none of the 13 benign lesions. All 6 hypoechoic lesions in the early phase were hemangiosarcoma. Hypoechoic pattern was significantly associated with malignancy (P= .02) with sensitivity of 38% (95% CI, 25–38%) and specificity of 100% (95% CI, 84–100%). An isoechoic pattern was found in 7 of the 16 malignant lesions and 10 of the 13 benign lesions. A heteroechoic pattern was found in 3 of the 16 malignant and 3 of the 13 benign lesions. Isoechoic and heteroechoic patterns were not associated with malignancy or benignancy.
In the late vascular phase, a hypoechoic pattern was found in 13 of the 16 malignant lesions and 2 of the 13 benign lesions. Hypoechoic pattern in the late phase was significantly associated with malignancy (P= .001) with sensitivity of 81% (95% CI, 66–90%) and specificity of 85% (95% CI, 65–95%). An isoechoic pattern was found in 1 of the 16 malignant lesions and 9 of the 13 benign lesions. Isoechoic pattern was significantly associated with benignancy (P= .001) with sensitivity of 69% (95% CI, 51–76%) and specificity of 94% (95% CI, 79–99%). A heteroechoic pattern was detected in 2 of the 16 malignant lesions and 2 of the 13 benign lesions. Heteroechoic pattern was not associated with malignancy or benignancy.
In the parenchymal phase, a hypoechoic pattern was found in 15 of the 16 malignant lesions and 11 of the 13 benign lesions. A heteroechoic pattern was found in 1 malignant lesion and 1 benign lesion. An isoechoic pattern was found in 1 benign lesion. There was no significant difference between malignant and benign lesions in the parenchymal phase.
Nodular hyperplasia (n= 8) was the most common benign lesion in this study (Fig 1). In the early vascular phase, 7 of the 8 nodules were isoechoic to the surrounding normal parenchyma (Fig 1C). In 6 of the 8 dogs, nodules remained isoechoic in the late vascular phase (Fig 1D). In 2 of the 8 dogs, the nodules became hypoechoic in the late vascular phase. In the parenchymal phase, all nodules were hypoechoic (Fig 1E). In 2 dogs, splenic nodules were diagnosed as hematoma (Fig 2). In the early and late vascular phases, both nodules were heteroechoic (Fig 2C and D). In the parenchymal phase, both nodules became homogeneously hypoechoic (Fig 2E). In 2 dogs, splenic nodules were diagnosed as extramedurally hematopoiesis. Both nodules were isoechoic during the early and late vascular phases. In the parenchymal phase, 1 was heteroechoic and another was isoechoic. In 1 dog, splenic nodule was diagnosed as granuloma. In the early vascular phase, the nodule was isoechoic. It became hypoechoic during the late vascular and parenchymal phase.
Hemangiosarcoma (n= 8) was the most common malignant tumor in this study (Fig 3). In 6 of the 8 dogs, nodules were hypoechoic during the early and late vascular phases. In their nodules, tortuous or aberrant wide vasculature was enhanced but the entire nodules were not enhanced (Fig 3B–D). In the other 2 dogs, nodules were heteroechoic in the early and late vascular phases. Seven of the 8 nodules were homogeneously hypoechoic in the parenchymal phase (Fig 3E) and another was heteroechoic.
Seven of the 8 malignant nodules other than hemangiosarcoma were isoechoic in the early phase (Fig 4C). However, contrast enhancement in these 7 nodules rapidly decreased, and they became hypoechoic in the late vascular phase (Fig 4D). Finally, all 8 lesions became hypoechoic in the parenchymal phase (Fig 4E).
The results of our study suggest that evaluation of enhancement pattern in perflubutane microbubbles-enhanced ultrasonography has value in differentiating between malignant and benign splenic nodules in dogs with high accuracy. However, the method for differentiating benign and malignant focal splenic lesions was quite different from that for the liver.13,14
The main result of this study is that there is no significant difference between benign and malignant lesions in the parenchymal phase. This finding is contrary to those of perflubutane microbubbles-enhanced ultrasonography of the liver, in which hypoechogenicity in the parenchymal phase is suggestive of malignant tumors. Parenchymal enhancement in the liver after perflubutane microbubbles injection is because of the distribution of the microbubbles in the Kupffer cells.9 The filling defect during the parenchymal phase created by the hepatic malignant tumor is then because of a decrease in the number of Kupffer cells. Conversely, hepatic nodular hyperplasia, the most common benign focal liver lesion in dogs, shows contrast enhancement during the parenchymal phase because the nodules contain Kupffer cells.13,14,16 On the other hand, splenic nodular hyperplasia, which is formed by hyperplastic lymphoid cells,17 became hypoechoic during the parenchymal phase in our study. Although the precise mechanism of splenic parenchymal phase imaging is not fully determined, we speculate that the contrast defect during the parenchymal phase created by splenic nodular hyperplasia might be because of a decrease of splenic macrophages. Therefore the parenchymal phase imaging was not useful for differentiation between benign and malignant splenic lesions. In some cases, however, nodules that could not be visualized with conventional ultrasonography became clearly hypoechoic in the parenchymal phase. Therefore, the parenchymal phase imaging could be useful for the detection of focal splenic lesions.
Another important finding is that the detection of hypoechoic nodules in the late vascular phase of perflubutane microbubbles-enhanced ultrasonography is suggestive of malignancy. In contrast, detection of isoechoic nodules in the late vascular phase is suggestive of benign lesions. These findings agree with those of previous studies using the contrast agent sulphur hexafluoride microbubbles.5,6 Sulphur hexafluoride microbubbles is a second-generation contrast agent that has been characterized to have splenic uptake in humans, allowing for parenchymal phase imaging in the human spleen.18 In dogs, however, it was demonstrated that sulphur hexafluoride microbubbles allowed for vascular phase imaging but not for parenchymal phase imaging.19 The vascular phase imaging with sulphur hexafluoride microbubbles could differentiate benign and malignant focal splenic lesions based on the finding that malignant tumors were hypoechoic to the surrounding normal spleen parenchyma in the wash-out phase (30 seconds after injection of sulphur hexafluoride microbubbles).5,6 In the normal dogs and humans, which have a sinusoidal spleen, a large sieve-like vascular reservoir is formed by an interconnected network of splenic sinusoids and red pulp spaces.17 It has been suggested that some contrast agents are pooled in the splenic sinusoids for a time after intravenous injection.20 Therefore, we speculate that the lack of normal sinusoids combined with neoplastic angiogenesis might be one of the causes of a malignant hypoechoic pattern during the late vascular phase.
The early vascular phase could also differentiate malignant and benign lesions with high specificity. Among them, hemangiosarcoma showed characteristic hypoechoic pattern during the early vascular phase. This finding concurred with those for sulphur hexafluoride microbubbles-enhanced ultrasonography5,6 and contrast-enhanced computed tomography.21 This hypoechoic area may correspond to the hemorrhagic or necrotic areas commonly associated with hemangiosarcoma. However, differentiation between hemangiosarcoma and hematoma should be done cautiously. In a previous study with the contrast agent perflutren lipid microsphere, hemangiosarcoma and hematoma showed similar heteroechoic patterns during the peak enhancement.7 In our study, likewise, 2 of the 8 hemangiosarcomas and both hematomas had similar heteroechoic patterns during the early vascular phase. Moreover, it was demonstrated that some cases of hematoma exhibited a hypoechoic pattern with sulphur hexafluoride microbubbles.5,6 Although the exact reasons for these differences are uncertain, we speculate that they might be because of the differences of contrast agents or patient populations in each study. Further studies are needed to clarify the criteria for discrimination between hemangiosarcoma and hematoma.
Evaluation of vessel appearance has no value in differentiating between malignant tumors and benign nodules. However, although some hemangiosarcomas had aberrant wide vessels, none of the other malignant or benign lesions, including hematoma, had such vessels. This finding agreed with that of a previous study.5 It is quite difficult to evaluate the vessel pattern accurately because of patient motion and the quite short duration of vascular enhancement in dogs. Vasculature pattern of focal splenic lesions should be evaluated cautiously but further studies are needed to evaluate its diagnostic significance.
aDaiichi-Sankyo, Tokyo, Japan
bAplio XG, Toshiba Medical Systems, Tochigi, Japan
cPLT-704 AT, Toshiba Medical Systems
dPSK-375 BT, Toshiba Medical Systems
eStatMate, ATMS, Tokyo, Japan
This study was funded partly by Research Fellowships of the Japanese Society for the Promotion of Science.