USE OF CONTRAST-ENHANCED ULTRASOUND FOR CHARACTERIZATION OF FOCAL SPLENIC LESIONS

USE OF CONTRAST-ENHANCED ULTRASOUND FOR

CHARACTERIZATION OF FOCAL SPLENIC LESIONS

FEDERICA ROSSI, VITO FERDINANDO LEONE, MASSIMO VIGNOLI, ETSER LADDAGA, ROSSELLA TERRAGNI

Contrast-enhanced ultrasound was used to study focal and multifocal lesions of the spleen in 26 dogs and two

cats affected by 11 benign and 18 malignant splenic diseases. A second-generation microbubble contrast medium

(Sonovue) was injected into the cephalic vein and enhancement patterns were subjectively described and time

intensity curves calculated. Final diagnosis was obtained by histopathologic examination after splenectomy

(n¼ 19) or by needle aspiration and sonographic follow-up after 4 and 8 weeks (n¼ 9). Contrast-enhanced

ultrasound parameters, improving the characterization between benign and malignant lesions, were established.

The most useful criterion was the hypoechogenicity of the lesion in the wash-out phase combined with the

presence of tortuous feeding vessels, which was observed in association with malignancy. All malignant lesions

were hypoechoic to the surrounding spleen 30 s after starting the contrast medium injection. Lymphosarcoma

and hemangiosarcoma had characteristic perfusion patterns. Lymphosarcoma had rapid time to peak and early

wash-out phase with a honeycomb pattern during the wash-out. Hemangiosarcomas were large nonperfused

masses in all phases surrounded by hypervascular splenic parenchyma. Benign lesions except one hematoma and

a benign histiocytoma had the same perfusion pattern as the surrounding spleen. Ultrasonographic and contrast-

enhanced ultrasound findings of an accessory spleen are reported. Contrast-enhanced ultrasound can improve

the characterization of focal or multifocal lesions of the spleen. Veterinary Radiology & Ultrasound, Vol. 49,

No. 2, 2008, pp 154–164.

Key words: contrast-enhanced ultrasound, microbubble contrast medium, small animals, spleen, sulfur

hexafluoride.

Introduction

FOCAL SPLENIC LESIONS are frequently identified during

routine ultrasonographic examination, especially in

old animals. They are often incidental findings and repre-

sent a diagnostic challenge. Because the echo pattern and

large vascular architecture of benign and malignant con-

ditions of the spleen are similar,1–5 baseline gray-scale and

color Doppler ultrasound have limited accuracy in char-

acterizing splenic disorders. Therefore, cytology or histol-

ogy are required for the final diagnosis.

Contrast-enhanced ultrasound involves injecting a small

quantity of a microbubble contrast medium into a periph-

eral vein with subsequent sonographic enhancement of a

vessel or tissue. With second generation contrast media, the

nonlinear oscillation of the microbubbles can be detected in

real time, and the perfusion of parenchymal focal lesions

can be compared with the surrounding tissue.

In Europe, contrast-enhanced ultrasound is routinely

used in humans to characterize liver lesions and the diag-

nostic accuracy is similar to that of contrast-enhanced

computed tomography (CT) and magnetic resonance (MR)

imaging.6–8 Recently, abdominal extrahepatic application

has been proposed.9,10 Initial studies suggest that contrast-

enhanced ultrasound is of diagnostic value in patients with

splenic and perisplenic lesions,11–16 but little information is

available on the perfusion pattern of focal splenic nodules.

Benign neoplasia is uncommon in humans and malignancy

is extremely rare. Lymphoma and angiosarcoma are re-

ported only occasionally,17,18 nodular hyperplasia is a rare

condition with a postmortem incidence ranging from

0.024% to 0.13%.19

In small animals, contrast-enhanced ultrasound has

been used to study normal liver perfusion,20, 21 liver focal

lesions,22 portosystemic shunts,23 and lymphadenomegaly

caused by lymphoma.24 This method can be used in small

animals without adverse effects and it is useful in charac-

terizing liver nodules and in identifying the vascular archi-

tecture of malignant lymph nodes.

The aim of this prospective study was to evaluate

whether contrast-enhanced ultrasound can be used to more

accurately characterize the perfusion of splenic focal ab-

normalities in small animals. Moreover, we attempted to

establish criteria that can be used to distinguish benign

from malignant splenic lesions and different types of

malignancies.

Address correspondence and reprint requests to F. Rossi, at the aboveaddress. E-mail: [email protected]

Received April 20, 2007; accepted for publication October 4, 2007.doi: 10.1111/j.1740-8261.2008.00343.x

From the Veterinary Clinic dell’Orologio–Via Gramsci 1/4 - 40037Sasso Marconi, Bologna, Italy (Rossi, Leone, Vignoli, Terragni), DVL,Veterinary Laboratory, Via Gramsci 1/3–40037 Sasso Marconi, Bologna,Italy (Laddaga).

154

Materials and Methods

Dogs and cats were included in this study according

to the following criteria: (1) single focal or multifocal

sonographic splenic lesions identified during routine sono-

graphy, (2) it was possible to perform the contrast-

enhanced ultrasound study without sedation or anesthe-

sia, and (3) splenectomy with subsequent histopathologic

examination, or diagnostic cytology was performed.

After the initial gray-scale ultrasound study, a 20G

intravenous cannula was placed in the cephalic vein. Saline

was administered during the procedure. Animals were

maintained in right lateral recumbency without sedation or

anesthesia and, during the examination, heart, and breath-

ing rate, femoral pulse, and mucosal refilling time were

recorded.

The ultrasound equipment included two systems with

coded harmonic capabilities� with two probes, a curvilin-

ear transducer with a receive frequency of 3.2MHz and a

linear probe with a receive frequency of 5MHz. The first

system was used for the first 21 dogs. The last nine dogs

were examined with the other equipment, which became

available during the second part of the study. For the

contrast study, the two machines functioned with the same

detection mode (contrast-tuned imaging mode). The same

two probes were used during the entire study. To reduce

the acoustic impact of the ultrasound waves on the micro-

bubble contrast agent, and to increase the persistence of the

contrast medium in the blood, the mechanical index was

always lower than 0.1 (range 0.05–0.1), which corresponds

to an acoustic pressure lower than 45kPa. A single focal

zone was placed in the deepest part of the lesion. The

overall gain and time-gain compensation were set so that

no signal from the underlying parenchyma of the spleen

was present; only very low background signal from the

splenic capsule or perisplenic vessels were maintained to

have an anatomic reference in the image. Standard and

contrast-enhanced ultrasound examinations were per-

formed by the same operator (F.R.). Part of the normal

splenic parenchyma surrounding the lesion was included in

the image so that it was possible to compare flow in two

different areas. A bolus of a second-generation sulfur

hexafluoride contrast mediumw was injected intravenously

at a dosage of 0.03ml/kg of prepared solution (5mg/ml). A

5ml normal saline flush followed. The timer was activated

at the moment of starting the injection and the flow of the

contrast medium into the lesion was observed in real time.

Care was taken to keep the probe in exactly the same po-

sition for at least 2min. Each animal received two injec-

tions of contrast medium. During the first injection the

total gain, time-gain compensation and position of the fo-

cal zone were occasionally changed to optimize the image,

and this setting was then maintained unchanged for the

subsequent injection. The second injection was performed

at about 5min following the first. Between the two injec-

tions, the ultrasound system was reset to its fundamental

setting and the cranial abdomen was scanned so that re-

sidual microbubbles could be destroyed before the second

examination.

In patients where there was more than one splenic lesion

with a similar baseline sonographic appearance, the largest

and more clearly imaged lesion was evaluated first. The

entire procedure was digitally recorded for later review.

Recorded movies were subjectively reviewed and a group

consensus formulated to describe the perfusion of the le-

sions and surrounding spleen.

The lesions were analyzed as follows. Echogenicity dur-

ing the wash-in, peak, and wash-out phases was defined as

hyper-, iso-, or hypoechoic compared with normal sur-

rounding spleen. Contrast enhancement in the lesion was

classified as absent (no enhancement), rim-like (continuous

ring of uniform peripheral enhancement), peripherally

irregular (with a peripheral rim of enhancement of vari-

able thickness), honeycomb (small hypoechoic areas),

diffusely homogeneous, and diffusely heterogeneous

(Table 1). Afferent vessels were described as either peri-

pheral or central, and straight or tortuous.

A commercial softwarez application was used to mea-

sure the mean pixel value in a region of interest. Because

any change in the setting parameters modifies the results of

a quantitative analysis, only the videos acquired during the

second injection were used for this evaluation. A frame was

selected every 2 s for the first 2min of the videos. In each

sampled frame, two different regions of interest were

drawn, inside the lesion and in the normal splenic paren-

chyma, at approximately the same depth. Inside homoge-

neous lesions, one region of interest (as large as possible)

was depicted in the center, whereas inhomogeneous chang-

es were sampled by two or more regions of interest, rep-

resenting parts with different perfusion.

Time–intensity curves were constructed using commer-

cial software.y The peak intensity, time to peak, average

up- and down-slope of the lesions and surrounding splenic

parenchyma were calculated. Time to peak was measured

from T0 (time of injection). To calculate average up- and

down-slope, the part of the curve with values above 10% of

baseline and up to about 90% of the peak enhancement

was used.

Nineteen dogs underwent splenectomy and the final

diagnosis was obtained upon histologic analysis of the

surgical specimen. In the remaining nine patients (seven

dogs and two cats), needle aspiration of the lesion was

�CnTI Megas Esatune, Esaote Firenze, Italy CnTI Mylab 30, EsaoteFirenze, Italy.wSonovue

s

, Bracco, Milano, Italy.zNIH Image J (US National Institute of Health, Bethesda, MD).yMicrosoft Excel 2000, Microsoft Corporation, Richmond, WA.

155CONTRAST-ENHANCED ULTRASOUND OF SPLENIC LESIONSVol. 49, No. 2

performed, followed by cytology. Because a diagnosis

based on cytologic sampling can be incorrect,25 ultrasound

examinations were performed again after 4 and 8 weeks to

reassess the sonographic features. Number, size, and echo-

genicity of the splenic nodules were compared with the

findings observed at the time of the contrast study.

Results

Between October 2003 and December 2006, 26 dogs and

two cats were studied. There were 14 males (13 dogs and

one cat) and 14 females (13 dogs and one cat). Mean age

was 10.0� 2.7 years (standard deviation [SD]) with a range

from 2.6 to 15 years.

On gray-scale ultrasound examination, single (n¼ 19) or

multiple parenchymal lesions (n¼ 9) of variable size and

echogenicity were detected (Table 1). The final diagnosis

included 11 benign and 18 malignant splenic lesions. One

dog had two different conditions, nodular hyperplasia and

accessory spleen. The 11 benign lesions included nodular

hyperplasia (n¼ 6), extramedullary hematopoiesis (n¼ 2),

hematoma (n¼ 1), benign fibrous histiocytoma (n¼ 1), and

one accessory spleen (splanunculus) (n¼ 1). Malignancy

was represented by the following diseases: lymphosarcoma

(n¼ 7), hemangiosarcoma (n¼ 4), malignant fibrous hist-

iocytoma (n¼ 2), undifferentiated sarcoma (n¼ 1), hist-

iocytic sarcoma (n¼ 1), liposarcoma (n¼ 1), mast cell

tumor (n¼ 1) and metastasis (n¼ 1).

The entire ultrasound procedure was always performed

in o10min and there were no changes in the monitored

physiologic parameters.

Small and superficial lesions (n¼ 19) were examined with

the 5MHz linear probe only, whereas only the 3.2MHz

curvilinear probe was used for large and deep nodules or

masses (n¼ 10). When possible (n¼ 3), the same lesion was

studied with both transducers and the two contrast se-

quences were compared. In these three patients, no differ-

ence was found in the perfusion pattern. With the linear

probe, image spatial resolution was subjectively judged to

be higher for both the fundamental and the contrast study.

During the first injection, the parameters were some-

times adjusted slightly, so that they were set optimally for

the second injection. This was necessary primarily for small

and superficial nodules, where the time gain compensation

in the near field was initially too low according to the

technical suggestions delivered from the experience in

human imaging.

The flow of the contrast medium in the abnormal and

surrounding normal spleen was easily detected and com-

pared in all patients by subjective analysis. The quantita-

tive analysis and construction of the perfusion curves

helped to better describe the perfusion patterns primarily

in the first seconds of the examination, during the wash-in

phase, when the changes in echogenicity were sometimes

very fast. Moreover, additional information was obtained

in the hematoma, where the quantitative analysis enabled

identification of low perfusion, which was difficult to detect

with subjective analysis.

In the fundamental study, the benign lesions were of

various size and echogenicity (see Table 1). In nine of the

11 benign lesions (nodular hyperplasia, extramedullary he-

matopoiesis, and accessory spleen), the lesions had similar

wash-in and wash-out to the surrounding spleen, so that

after a few seconds the nodules were isoechoic and diffusely

homogeneous. In this group, only two small hyperplastic

nodules were briefly hyperechoic in the early arterial phase,

but at peak and in the wash-out phase they had the

same echogenicity as the surrounding normal spleen (Fig.

1A–D). No feeding vessels were evident. The accessory

spleen was a round hilar structure (about 2 cm in diame-

ter), homogeneous, and isoechoic to the spleen. With

contrast-enhanced ultrasound, the perfusion of the acces-

sory spleen was identical to that of the adjacent spleen

(Fig. 2A and B). In the nine benign lesions, mean time to

Table 1. Histological Type and Gray-Scale Appearance of Splenic Focal Lesions

LesionMean Size

(Range) (cm)Single (s)

or Multiple (m) Hypoechoic Hyperechoic Isoechoic Mixed Complex Target

Malignancy (n¼ 18)

LSA (n¼ 6) 2.1 (0.7–3) 5 sþ 1m 5 1HSA (n¼ 4) s 1 1 2Malignant fibrous histiocytoma (n¼ 2) 9 (8–10) s 1 1Undifferentiated sarcoma (n¼ 1) 6 s 1Histiocytic sarcoma (n¼ 1) 1.6 s 1Liposarcoma (n¼ 1) 4 s 1Mast cell tumour (n¼ 1) o1 m 6Metastasis (n¼ 1) o1 m 3Benignancy (n¼ 11)

Nodular hyperplasia (n¼ 6) 1.1 (0.3–2) 2 sþ 4m 3 4 2 1Extramedullary hematopoiesis (n¼ 2) o1 m 1 1Hematoma (n¼ 1) 0.8 s 1Benign fibrous histiocytoma (n¼ 1) 3.5 s 1Accessory spleen (n¼ 1) 1 s 1

156 ROSSI ET AL. 2008

peak was 34.4 s (SD 6.6) for lesions and 34.4 (SD 8.3) for

surrounding normal spleen. Mean lesion peak intensity was

93.7% of the surrounding normal spleen (SD 10.6%).

Mean up-slope was 3.5 mean pixel value/s (SD 0.6) for the

benign lesions and 3.1 mean pixel values/s (SD 1.0) for the

surrounding normal spleen. Mean down-slope was �0.89mean pixel value/s (SD 1.0) for the benign lesions and

�0.58 mean pixel values/s (SD 0.6) for the surrounding

normal spleen.

The hematoma (Fig. 3) appeared as a small (0.8 cm)

target lesion in gray-scale imaging. After contrast medium

injection, it was a round homogeneous hypoechoic area

with constant, low homogeneous perfusion. Lesion mean

pixel value intensity, measured from the initial rise of the

curve (12 s) to the end of the examination, was 22% of the

peak intensity measured in the surrounding spleen (range

15–27%). The benign histiocytoma, a 3.5 cm hypoechoic

nodule in baseline imaging, was the only clearly hyperper-

fused lesion. Compared with the surrounding parenchyma,

it had rapid, intense enhancement and was strongly hype-

rechoic in the wash-in phase. After the peak, the echo-

genicity decreased slowly and the lesion was isoechoic to

the surrounding normal spleen after 50 s. In the first few

seconds after contrast medium injection, multiple small

vessels were seen, homogeneously distributed in the central

part of the lesion. Time to peak intensity was 16 s, peak

intensity was more than 2.5 times the peak intensity of the

surrounding normal spleen (268%). Up- and down-slope

of the lesions were 12.8 and �1.5 mean pixel value/s, the

up- and down-slope of the surrounding spleen were 2.0 and

�0.6 mean pixel value/s.

In all malignant lesions, the perfusion was very different

in comparison to that in normal splenic parenchyma. After

contrast medium administration, lesion size and margins

were better defined because of higher contrast resolution of

the adjacent spleen. In three patients (two lymphosarcoma

and one hemangiosarcoma) additional smaller nodules

which had not been noted in the fundamental study were

detected. These nodules were small (mean size 0.4 cm) and

isoechoic to the surrounding normal spleen in gray-scale

imaging. With contrast-enhanced ultrasound, they were

easily detected due to different echogenicities. Malignant

lesions had variable echogenicity in the wash-in phase and

at peak. All malignant lesions became either completely or

Fig. 1. Gray-scale (A) and contrast enhanced ultrasound (B–D) images of a hyperplastic splenic nodule. The lesion is a round hypoechoic nodule infundamental ultrasound (A). In the first seconds of the contrast-enhanced ultrasound examination (B), before the entrance of the contrast medium, the image isblack because of the suppression of the fundamental signal. After 13 s (C), the nodule is slightly hyperechoic to the surrounding spleen, but it becomescompletely homogeneous after a few more seconds (D).


extensively hypoperfused during the wash-out phase. In 12

patients (lymphosarcoma, malignant fibrous histiocytoma,

histiocytic sarcoma, mast cell tumor, metastasis), the wash-

out was fast and homogeneous. In the remaining six pa-

tients (hemangiosarcoma, undifferentiated sarcoma, and

liposarcoma), the wash-out was inhomogeneous with large

hypoperfused areas in the late phase. Table 2 summarizes

the observed perfusion patterns.

Excluding the hemangiosarcoma and the undifferentiat-

ed sarcoma, which had no peak, all other malignant lesions

had a mean time to peak of 23 s (SD 7.8). All malignant

lesions were hypoechoic to the surrounding spleen 30 s

after starting the injection.

Hemangiosarcoma (n¼ 4) and the undifferentiated

sarcoma (n¼ 1) were characterized in all phases by homo-

geneous anechoic (nonperfused) areas with highly

vascularized surrounding parenchyma (peripheral irregu-

lar perfusion pattern). Thin septae of hyperechoic tissue

entered the anechoic areas with a sharp demarcation be-

tween these two different parts. Tortuous vessels were vis-

ible on the periphery of the lesion (Fig. 4). Based on the

time–intensity curves, there was no enhancement in the

large anechoic lesions, and pixel intensity did not change

from the baseline.

Lymphosarcoma (n¼ 7) and histiocytic sarcoma (n¼ 1)

were isoechoic (n¼ 6) or hyperechoic (n¼ 2) in the wash-in

phase, with a diffuse homogeneous enhancement and a

rapid time to peak. At peak, they were hypoechoic (n¼ 6)

or hyperechoic (n¼ 2) to the surrounding spleen. The le-

sion wash-out phase started before the splenic peak (early

wash-out phase) and all nodules became hypoechoic dur-

ing this phase. The contrast medium washed out progres-

sively from the lesions, leaving a round hypoechoic lesion

characterized by small round anechoic areas (honeycomb

Fig. 3. Gray-scale (A) and contrast enhanced ultrasound (B) images of a hematoma, appearing as small (0.8 cm) target lesion in gray-scale imaging. Aftercontrast medium injection, it appeared as a round homogeneous hypoechoic area with constant, low homogeneous perfusion.

Fig. 2. Accessory spleen in a 9-year-old female Boxer. A round structure, homogeneous and isoechoic to the spleen, is visible at the splenic hilus (A). Withcontrast-enhanced ultrasound the perfusion of the accessory spleen is identical to the adjacent normal spleen (B). This enables differentiation between theaccessory spleen and a splenic lymph node.


Table2.Contrast

EnhancedUltrasoundDiagnostic

Findings/CriteriaforMalignancy

andBenignancy

Contrast

Enhan

cedUltrasoundDiagnostic

Findings/criteria

forMalignancy

andBenignancy

Lesion

Contrast

EnhancedUltrasoundFindings

Echogenicity(lesionvs.spleen)

EnhancementPattern

FeedingVessels

Wash-in

Peak

Wash-out

Malignancy

(n¼18)

LSA

(n¼7)

Hyper

(n¼2)–iso(n¼5)

Hyp

er(n¼2),

hypo(n¼5)

Hypo

Diffuse

homogeneous(w

ash-in,peak),

honeycomb(w

ash-out)

Finenet

ofthin

uniform

lydistributed

vesselin

thelesion

Hystiociticsarcoma(n¼1)

Iso

Hyp

oHypo

Diffuse

homogeneous(w

ash-in,peak),

honeycomb(w

ash-out)

Finenet

ofthin

uniform

lydistributed

vesselin

thelesion

HAS(n¼4)þ

Hyper

(periphery),

hypo(center)

Hyp

oHypo

Peripheralirregu

lar,absence

of

enhan

cementin

thehyp

oechoicareas

Tortuousvesselsattheperiphery

Undifferentiatedsarcoma(n¼1)

Hypo

Hyp

oHypo

Peripheralirregu

lar,absence

of

enhan

cementin

thehyp

oechoicareas


Malignantfibroushistiocytoma(n¼2)

Hypo

Hyp

oHypo

Diffuse

homogeneous


Metastasis(n¼1)

Hypo

Hyp

oHypo

Diffuse

heterogeneous(w

ash-in),

diffuse

homogeneous(peak,wash-

out)

Thin

tortuousvesselsaroundthenodules

Mast

celltumor(n¼1)

Hypo

Hyp

oHypo

Diffuse

heterogeneous(w

ash-in),

diffuse

homogeneous(peak,wash-

out)

Thin

tortuousvesselsin

theparenchym

a

Liposarcoma(n¼1)

Hyper

Iso

Hypo(centre),

iso(periphery)

Peripheralirregu

lar

Tortuousvesselsat

theperiphery

Benignancy

(n¼11)

Nodularhyperplasiaandextramedullary

hem

atopoiesis(n¼8)

Iso(n¼6)slightlyhyper-(n¼2)

Iso-

Iso

Diffuse

homogeneous

Sim

ilarto

thesurroundingspleen

Hem

atoma(n¼1)

Hypo

Hyp

oHypo

Diffuse

homogeneous,mild

enhan

cement

Novessels

Benignhistiocytoma(n¼1)

Clearlyhyper-

Hyp

er-

Hyper-

Diffuse

homogeneous(w

ash-in),

diffuse

heterogeneouswithring-

enhan

cement(w

ash-out)

Dense

vesselsdistributionin

thecentral

part

Accessory

spleen

(n¼1)

Iso

Iso

Iso

Diffuse

homogeneous

Sim

ilarto

thesurroundingspleen


pattern). Feeding vessels were represented by a fine net of

thin, uniformly distributed vessel (Fig. 5).

Mean time to peak was 21.7 s (SD 4.9) for the lesions

and 36 s (SD 9.6) for surrounding spleen. Mean peak in-

tensity was 92.7% (SD 25.6%) of the mean surrounding

spleen peak intensity. Mean up-slope was 10.1 mean pixel

values (SD 2.0) for the malignant lesions and 5.0 mean

pixel values (SD 2.5) for surrounding normal spleen. Mean

down-slope was �2.0 mean pixel values (SD 0.4) for the

lesions and �0.92 mean pixel values (0.3 SD) for the sur-

rounding normal spleen.

All nine lesions diagnosed with cytology (nodular hy-

perplasia, extramedullary hematopoiesis, and the accessory

spleen) were benign. Based on the sonographic follow-up

examination after 4 and 8 weeks there were no changes in

number, size, or echogenicity of the nodules.

Discussion

Sonovue is a second generation ultrasound contrast me-

dium containing a central gaseous nucleus (sulfur hexaflu-

oride) stabilized by an external elastic phospholipidic shell.

The low solubility of sulfur hexafluoride improves its

stability in the vascular compartment. These very small

microbubbles (mean diameter 2.5mm) are able to pass the

pulmonary capillary circulation and remain in the blood

pool for a longer time compared with first-generation

contrast media like Levovist. For this reason, sulfur hexa-

fluoride can be used both as a macrovascular and a

microvascular enhancer.

When exposed to an ultrasound wave, microbubbles

have significantly higher reflectivity than blood and soft

tissues,26 so they enhance the acoustic signal in the blood

circulation. When imaged with an ultrasound wave of low

acoustic power (mechanical index lower than 0.1 or

45kPa), the microbubbles are minimally destructed and

oscillate continuously, allowing real-time imaging of tissue

perfusion. When compared with the incident wave, the

reflected ultrasound is modified in two ways:27 First, it

contains nonlinear components, as a consequence of the

asymmetric oscillation of the microbubbles. Second, it in-

cludes harmonic frequencies. To improve the contrast-to-

tissue ratio and maximize the distinction between blood-

derived signal and tissue-derived signal, contrast-specific

Fig. 4. Contrast-enhanced ultrasound examination (A–C) and time–intensity curve (D) of a large complex mass in a dog with hemoperitoneum (finaldiagnosis: hemangiosarcoma). In the first few seconds, tortuous vessels are seen at the periphery of the lesion (A). The lesion has a peripheral irregular perfusionpattern, with a homogeneous anechoic (nonperfused) central area surrounded by highly vascularized parenchyma (B). Smaller anechoic nodules are visible (C).The enhanced parenchyma on the left of the images is the caudal left margin of the liver. (D) Time–intensity curve. Triangle line, region of interest in the centreof the lesion has no enhancement; dotted line, region of interest in the surrounding spleen.


imaging modalities were developed. Because the difference

in microbubble tissue backscatter is much higher for har-

monic frequencies, harmonic imaging can be used as a

contrast-specific method to clearly depict differences in the

perfusion of tissues. The flow of contrast medium can be

assessed by coded harmonic imaging, - that is, by sup-

pressing the contribution of the fundamental frequencies in

the image construction. Another way to distinguish the

contrast-specific signal from the tissue-derived signal is to

detect nonlinear components by pulse-cancellation tech-

niques. With this modality, multi-pulse sequences with

precise changes in transmitted inter-pulse amplitude and

phases are used to reject the linear fundamental tissue sig-

nal and retain the nonlinear signal from the microbubbles.

If precise, this modulation produces very good contrast-to-

tissue ratio and has high sensibility in the detection of the

contrast medium signal.28

In this study, coded harmonic imaging was used to

evaluate the perfusion of focal splenic lesions in a popu-

lation of 26 dogs and two cats of various ages. The flow of

contrast medium could be visualized easily in the vascular

part of the lesion and in the surrounding normal splenic

parenchyma in the real-time examination. The linear

5MHz probe was adequate for small and superficial le-

sions, whereas the 3.2MHz convex probe was necessary for

examining large and deeper masses and for screening the

liver. In patients where both probes were used, no differ-

ence was found regarding the analysis of perfusion; how-

ever, the study quality was subjectively judged to be

superior with the linear probe because of its higher spatial

resolution.

All examinations were performed without sedation or

anesthesia, and the animals were easily restrained in right

lateral recumbency for the time required for the study,

which was always o10min. The injection of contrast me-

dium did not produce any physiologic changes, supporting

that the procedure is safe and well tolerated. Noncooper-

ative animals, mainly cats, may need to be sedated or

anesthetized, and possible secondary changes in splenic

blood flow must be considered. It is known that anesthetic

drugs such as acepromazine, thiopental and medetomi-

dine–diazepam–ketamine can increase splenic volume in

Fig. 5. Contrast enhanced ultrasound examination (A–C) and time–intensity curve (D) of a dog with a splenic lymphosarcoma. During the wash-in phase(A), the nodule is slightly hyperechoic with a diffuse homogeneous enhancement. In the wash-out phase (B), the lesion becomes hypoechoic with small anechoicround areas (honeycomb pattern). A second smaller nodule is visible on the left. This was not detected in the fundamental examination. (D) Time–intensityCurve. Pink line, region of interest in the center of the lesion; blue line, region of interest in the surrounding normal spleen.


dogs.29,30 Smooth muscle relaxation has been hypothetized

to produce passive congestion during anesthesia and in this

situation the presence of microbubbles in the red pulp si-

nusoids would probably increase. It has been reported that

propofol does not modify splenic size, whereas it shortens

the time to peak enhancement of the liver in normal dogs

after Sonovue administration.21 No studies have yet been

performed to quantify the perfusion rate of the spleen in

anesthetized animals. It would be necessary to compare the

same animals before and after sedation or anesthesia to

establish whether these drugs modify the normal splenic

enhancement and the appearance of focal lesions during

the contrast study.

In this study, 29 focal or multi-focal splenic lesions in 26

dogs and two cats were examined with contrast-enhanced

ultrasound. Subjective and quantitative analysis of the vid-

eo enabled the perfusion of the lesions to be described

during the wash-in, peak and wash-out phases, comparing

the focal changes with the surrounding normal spleen.

Sonographic findings were compared and grouped accord-

ing to the final diagnosis, with the aim of establishing cri-

teria that could be associated with benign and malignant

splenic lesions (Table 1). In this series, most benign lesions

(10/12) had a perfusion pattern similar to the adjacent

parenchyma, so that the lesions were isoechoic to the

surrounding normal spleen after the first few seconds. All

these lesions were homogeneously isoechoic in the wash-

out phase. This can be explained by the similar architecture

of the vascular network associated with benign hyperplas-

tic conditions and normal spleen.

The hematoma was the only benign-hypoperfused lesion

and it had very mild enhancement, which could represent

revascularization after hemorrhage. Unfortunately, this

was a single lesion and nothing can be assessed about the

perfusion pattern in hematomas of different ages. Absence

of associated hyperperfused areas and tortuous feeding

vessels were recognized as criteria for differentiating this

hematoma from the hypovascular areas observed in all

hemangiosarcomas. In this comparison, the quantitative

analysis added another differential criterion of a low but

constant perfusion in the hematoma vs. a complete absence

of any enhancement in the large hypoechoic areas associ-

ated with the hemangiosarcomas. This single observation

agrees with a recent CT study, in which splenic hematomas

were characterized by higher attenuation in comparison

with hemangiosarcomas after contrast medium adminis-

tration.31 When comparing these results, it must be con-

sidered that the enhancement mechanism is different in CT

vs. contrast-enhanced ultrasound, because the iodinated

contrast medium diffuses in the extracellular space whereas

the microbubble contrast medium is a pure blood-pool

agent. Despite this difference, sonographic and CT features

of splenic lesions overlap in humans.11,32 Spontaneous

splenic hemorrhage is an uncommon condition in dogs;

however, hematomas inside nodular hyperplasia lesions oc-

cur frequently and are believed to represent a continuum.33

Therefore, areas of low uptake associated with benign hy-

perplasia may be expected with contrast-enhanced ultra-

sound. Even though the examination of this hematoma

suggests that contrast-enhanced ultrasound could be helpful

in differentiating this type of lesion from hemangiosarcoma,

no conclusion can be made from a single patient.

In this study, we also identified a hilar accessory spleen

in a dog. Accessory spleens are frequently reported as in-

cidental sonographic findings in humans. These single or

multiple islands of normal splenic parenchyma occur fre-

quently at the splenic hilum, and they may also be found in

the gastrosplenic and splenorenal ligaments or in the tail of

the pancreas, but rarely elsewhere in the abdomen.34 Mi-

crobubble contrast medium helps distinguish them from an

enlarged hilar lymph node or a pancreatic mass.11,15 The

accessory spleen has enhancement identical to that of the

parent spleen and retains contrast medium in the paren-

chymal phase.32,33 Hilar lymph nodes have transient

enhancement in the arterial phase.12 In 14 human patients

with perisplenic lesions, six neoplastic conditions were

differentiated from eight accessory spleens because of the

contrast-enhanced ultrasound pattern.15

The perfusion pattern of malignant lesions was different

than observed in the surrounding normal parenchyma. Af-

ter variable wash-in and peak phases, all malignant lesions

became completely or extensively hypoperfused during the

wash-out phase. Thirty seconds after injection, these hypo-

echoic lesions were easily detected, as they were surrounded

by hyperechoic splenic parenchyma. Associated tortuous

vessels were observed in all malignant lesions. These results

confirm the observations reported for splenic malignancy in

humans9–11 and are analogous to the contrast-enhanced

sonographic features of liver neoplasia in small animals,22

suggesting that hepatic and splenic tumors behave similarly

with contrast-enhanced ultrasound, even though the nor-

mal vascular anatomy of these organs is different.

The vascular network of the spleen is complex and or-

ganized differently in the two interconnected compart-

ments, the white and red pulp. The white pulp contains

small pulpar arteries, branches of the trabecular arteries,

which originate in the splenic hilus and follow the subdi-

vision of the splenic trabeculae. The lymphatic follicles are

organized around these small arterioles. The red pulp is the

real blood reservoir. Most of the erythrocytes are con-

tained in large, thin-walled splenic sinusoids. The sinusoids

are supplied by the penicillar arterioles of the adjacent

splenic cords and continue into the pulpar and trabecular

veins. The presence of such a large vascular space with slow

blood flow is the most probable explanation for splenic

enhancement after injection of Sonovue. Even the site of

Sonovue accumulation has not been established, and

although true splenic tropism has been suggested,37 passive


pooling of microbubbles in the sinusoids seems to be the

most reliable hypothesis.12 We conclude that contrast-en-

hanced ultrasound allows detection of abnormal perfusion

patterns associated with splenic malignancies. We did not

identify why malignant lesions have a different type of per-

fusion compared with the normal parenchyma, the reasons

are probably very complex and different for the various

types of lesions. The large nonperfused areas seen with

hemangiosarcoma may correspond to the necrotic and

hemorrhagic areas associated with these tumours. Lympho-

sarcoma had early wash-in and wash-out phases, and this

possibly reflects the lack of normal sinusoidal vessels of the

red pulp combined with neoplastic angiogenesis.

Limitations of this study are the heterogeneity of the

lesions and the lack of other common types of neoplastic

and nonneoplastic splenic conditions. Many types of sar-

comas (mixo-, leiomio-, fibro-, osteosarcoma) were not

observed in this study, therefore, it is not known if they

meet our criteria for malignancy. Other neoplastic condi-

tions (liposarcoma, mast cell tumor, metastatic disease)

were seen only in isolated patients, so it is not possible to

verify whether they have specific perfusion patterns. Benign

conditions, like abscess, hemorrhage, or infarction, can

appear as focal solid lesions on gray-scale ultrasound and

are expected to be hypoperfused areas with contrast-en-

hanced ultrasound, similar to the hematoma. Limitations

of contrast-enhanced ultrasound in the differentiation be-

tween benign from malignant conditions of the spleen

could be related to the complexity of the splenic lesion. The

borderline between benign and malignant splenic condi-

tions can be difficult to define, even with histopathology,

because some lesions have transitional features.38

We suggest there is a potential role for contrast-en-

hanced ultrasound in the differentiation of focal splenic

lesions. A larger number of patients, including all types of

conditions, are needed to confirm our findings and to in-

crease knowledge in this complex field. Traumatic splenic

injuries and vascular disease are indications to perform a

contrast-enhanced ultrasound study of the spleen in hu-

mans,11–15 and these are also possible applications in vet-

erinary medicine. In animals with thromboembolism,

infarction due to partial splenic torsion or posttraumatic

splenic rupture, contrast enhanced ultrasound could be

useful in confirming the diagnosis.

In humans, contrast-enhanced CT and MR imaging are

gold standard diagnostic imaging methods for diagnosing

and characterizing focal lesions of the spleen, and specific

perfusion patterns are known.11,39,40 Compared with CT

and MR imaging, contrast-enhanced ultrasound has sim-

ilar diagnostic performance with some advantages, being

less expensive, portable, and rapidly performed, without

ionizing radiation exposure.10 Therefore, contrast-en-

hanced ultrasound is considered to be a valid alternative

to contrast CT and MR imaging.

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USE OF CONTRAST-ENHANCED ULTRASOUND FOR CHARACTERIZATION OF FOCAL SPLENIC LESIONS