Pancreatic Evaluation in Dogs using Different Ultrasonographic Techniques – Preliminary Results

Abstract The aim of this study is to describe the preliminary results on the accuracy of ultrasonographic techniques such as elastography, contrast enhanced ultrasound (CEUS) and Doppler in determining pancreatic changes. Twenty-five dogs, males and females, aged 1-14 years, were studied. Sixteen animals had no clinical signs of pancreatic disease (GS) and nine presented signs of pancreatic disorders (GD). All animals from GD presented sonographic changes in B-mode and qualitative elastography, with shear-wave velocity (SWV) higher (2.4±0.5m/s) in GD (p=0.014) than GS (1.9±0.3m/s) resulting in 78% sensitivity and 69% specificity in the identification of pancreatic changes. Regarding Doppler mode, no differences were observed between groups with color mapping or pulsed wave Doppler. The values obtained with CEUS did not differ between groups. Elastography is a promising technique for differential diagnosis of pancreatic changes because of its sensitivity and specificity, while the other techniques did not show diagnostic accuracy.


INTRODUCTION
Among pancreatic diseases, pancreatitis is the one that most affects the exocrine pancreas of dogs. The disease is characterized by nonspecifi c and intermittent gastrointestinal signs that may hinder the diagnosis [1,2], causing complications such as cell death, reduced blood supply and it might trigger systemic infl ammation, leading to pancreatic dysfunction with a high mortality rate [3,4]. One report mentions a case of pancreatic destruction in a dog, in which it was only possible to fi nd remnants of the parenchyma and infl ammatory cells under the microscope [2].
Mild pancreatitis is a disease with low morbidity and mortality that can be reversed, if rapidly diagnosed [5].
The diagnosis of pancreatitis is still challenging, especially in mild cases, where patients present discreet clinical signs. Routinely, serum activities of amylase and lipase are used for diagnosis, because they are indicators of pancreatic infl ammation, however, these assays have low specifi city and sensitivity in the identifi cation of pancreatitis in dogs and their usefulness in clinical practice is questionable [6]. Nowadays, rapid tests that measure the level of the specifi c pancreatic lipase in the blood are being used. Even though they have good sensitivity and specifi city in cases of acute pancreatitis, they may not detect subclinical phases or they might even provide false positive results [7] Defi nitive diagnosis is made by histopathological examination, however, its execution in the ante-mortem organ is rare [5]. Cytological examination by fi ne-needle aspiration was widely used in medicine, but nowadays its use is restricted and controversial. There are reports about secondary contamination that may occur in patients with pancreatic necrosis and the detection of the etiological agent becomes more diffi cult [8].
Computed tomography (CT) and Magnetic Resonance Imaging (MRI) are widely used in medicine, however, in veterinary medicine, they are not easily accessible and require general anesthesia [9].
Due to the diffi culty in concluding the diagnosis of pancreatitis, the variety of differential diagnoses of acute abdomen in dogs and lack of precise assays, ultrasonography is the method of choice for the evaluation of patients with suspected pancreatitis. Despite its sensitivity of 68% [1,6], the exam provides important information about changes in the pancreatic parenchyma, already identifi ed by other authors [5]. It is important to emphasize that a normal ultrasound examination does not rule out pancreatitis. If the disease is suspected, but there are no deviations in the fi rst exam, ultrasound should be repeated in a few days, as the severity increases and sonographic changes tend to appear with time [6].
Although diagnostic accuracy of B mode ultrasonography is acceptable, this method cannot distinguish infl ammation, necrosis and neoplasia [5], thus, the use of new noninvasive ultrasonographic techniques, such as elastography and contrast enhanced ultrasound (CEUS) could contribute in the detection of pancreatic lesions and help the diagnosis of the patients.
CEUS is a new advancement in imaging diagnosis in medicine and its application in veterinary medicine is constantly growing to assess organ perfusion, detect areas of necrosis and aid in the diagnosis of pancreatic neoplasms [4,9]. Elastography is an imaging modality that evaluates the stiffness of the organs. Studies performed with other tissues have shown a positive correlation between shear wave velocity and the stiffness of the tissue evaluated, thus, the faster the shear wave velocity, the stiffer the tissue [10].
The aim of this preliminary study was to evaluate accuracy of different ultrasonographic techniques (B mode, Doppler, CEUS and Elastography) in assessing pancreatic changes in dogs and to describe the fi ndings of each method in different lesions.

MATERIALS AND METHODS
The study was performed and approved by the Ethics Committee in the Use of Animals (protocol n. 007976/18). Twenty-fi ve dogs of different breeds, aged between 1-14 years and weighing between 5-29kg were selected for this study. Dogs were allocated in two groups: GS (n=16) healthy animals and GD (n=9) non-healthy animals, with suspicion of pancreatic disease. All dogs came from the clinical practice of the Veterinary Hospital of the same institution, informed and written consent were obtained from client-owned animals included in this study.
Animals were classifi ed based on the results of physical and clinical examination, hematological results and B mode ultrasonography. GS animals did not present any alterations in these tests and GD presented pain in the epigastric region, abdominal discomfort and hematological changes, such as leukocytosis with left shift.
For ultrasound examination, all animals fasted for 8-12 hours prior to the exam, so there would not be superposition of the gastrointestinal content on the pancreas. The abdomen was clipped and acoustic gel was applied to facilitate the procedure and promote more contact between the transducer and the skin. Patients were positioned in dorsal recumbency and during the scan they were shifted to lateral (right and left) recumbency, in order to locate all portions of the organ under study (pancreas).
Scanning was performed by a single operator, with nine years of experience, using ACUSON S2000 -SIEMENS (Siemens, Munich, Germany) and linear and convex multifrequency transducers (7.5 to 9.0MHz).
B mode scanning was performed prior to elastography, aiming to evaluate the entire pancreatic parenchyma of the animals in its different anatomical portions (right and left lobes and body) and also to assess their characteristics: echogenicity (in comparison to adjacent structures), echotexture (homogenous and heterogenous), contours (regular and irregular) and dimensions (normal, increased and reduced). To locate the right pancreatic lobe, the anatomical landmarks used were right kidney, descending duodenum (coursing through the right abdominal wall) and the pancreaticoduodenal vein, located parallel to the descending duodenum.
In this study, all techniques were standardized on the right pancreatic lobe, because in GS it was the portion that could be identifi ed in all animals. In GD, all portions were assessed, however, only the values of the right lobe were considered.
Elastography was performed with the same equipment, using the software for Acoustic Radiation Force Impulse (ARFI) qualitative characterization and quantifi cation (Virtual Touch Tissue Quantifi cation, 2D-SWE technique). The qualitative ARFI method resulted in colored images of the pancreatic tissue (elastogram) for the evaluation of tissue deformity (with or without deformation), in which lighter tones (bluish) represent more elastic tissues (soft) and darker tones (reddish) represent stiffer tissues (hard). Image quality was tested using the exhibition mode, in which greenish and homogenous images indicated high quality of the technique and yellowish and heterogenous images indicated low quality.
In the quantitative ARFI method, it was possible to measure shear wave velocity (SWV m/s). In this study, fi ve measurements of the right pancreatic lobe were obtained and mean values were calculated for statistical analysis.
Using Color Doppler, vascular characteristics of the parenchyma were evaluated. In patients with suspected pancreatic disease, it was verifi ed whether neovascularization was present. In order to obtain vascular indices of both healthy and diseased tissue, after identifi cation of the pancreaticoduodenal vein with color mapping, the sample volume was positioned in the central portion, then, Pulsed Doppler was activated, obtaining the results of resistivity index (RI) automatically by the equipment.
For microbubble contrast enhanced ultrasound (CEUS), contrast-specifi c software was used (CADENCE®, Siemens, Munich, Germany), with secondary harmonic imaging, pulse inversion technique and a 9.0MHz linear transducer. The images obtained were evaluated in a specifi c imaging software. The contrast agent (SonoVue®, Bracco, Milan, Italy), was administered in the dosage of 0.1 mL for each animal, using a venous catheter that was maintained in the cephalic vein, followed by administration of 5.0 mL of saline solution (NaCl 0.9%).
Video clips lasting 5 minutes after injection of the contrast were obtained and registered in the internal system storage and then, they were analyzed. This contrast exam defi ned parameters related to the fi lling (homogenous or heterogenous) of the organ by microbubbles. Additionally, the times of vascular fi lling were analyzed, since administration of the contrast in the bloodstream until the beginning of the organ perfusion (wash-in); peak of contrast (enhancement); and time of the contrast total output from the parenchyma (wash-out).
Statistical analysis was performed using the R software (RTM Foundation for Stastistical Computing, Vienna, Austria). Sonographic variables were compared between clinical classifi cations using Student's T-test. Subsequently, parameters that presented a signifi cant difference were submitted to a discriminant analysis using ROC curves and cutoff values (CV), sensitivity, specifi city and area under the curve were calculated using the logistic regression model. A signifi cance of 5% (p=0.05) was established for all tests.

RESULTS AND DISCUSSION
B mode ultrasonography and elastography were performed without diffi culties and intercurrences. However, Spectral Doppler evaluation was very diffi cult in six GS patients (37.5%), because of the narrow nature of the organ and its small evaluation window. In the GD group, this evaluation was not possible in three patients (33.3%), due to the relentlessness and abdominal discomfort of the patients. In CEUS, it was not possible to evaluate fi ve patients from GS (31.25%), due to the localization and dimension of the organ. These limiting factors of our study reduced the number of patients evaluated in the described techniques.
In B mode scanning of GS dogs, the mean thickness was 0.67cm and it was possible to observe the right pancreatic lobe of normal size, preserved echogenicity (isoechoic to the mesenteric fat and the adjacent hepatic lobe) homogenous echotexture and regular contours. The characteristics found in the pancreas from the GS group were compatible to a healthy tissue.
All nine sick patients (GD) showed nonspecifi c signs, such as anorexia, apathy, vomiting, diarrhea, "praying position", pain in the epigastric region and sonographic alterations such as changes in echogenicity, echotexture, dimensions and contours. In seven cases (77.8%) changes in the adjacent organs were observed, reinforcing the suspicion of pancreatic disease ( Table 1).
The sonographic alterations found in this study (reduced echogenicity, increased or normal size of the organ, heterogenous echotexture and adjacent changes, such as reactive mesentery, free fl uid in the abdomen or duodenitis) are images suggestive of acute pancreatitis, corroborating with several studies [4][5][6][7]11,12]. All alterations were noted in the right lobe, in some patients in the pancreatic body and only one patient presented alterations in all portions of the organ, which reinforces data from the literature that states that the right pancreatic lobe is more easily located in dogs [6].
Pancreatic edema and adjacent free fl uid were seen in two patients. This was also observed in other studies that describe acute pancreatitis with swollen parenchyma, however, in these cases, it is necessary to consider differential diagnosis such as portal hypertension and hypoalbuminemia [12].
No sonographic signs of neoplasia were seen in any patient. This might be because of the small number of patients in this study. Although Bailey and Page [13] reported that pancreatic neoplasms (exocrine and endocrine) are rare in veterinary medicine in comparison to human medicine, pancreatitis, in contrast, is routinely detected in the canine pancreas, as observed in the present study.
In the qualitative elastography, GS group presented homogenous pancreatic parenchyma and of bluish colors (deformable), being characterized as soft. In eight GD patients, the elastogram presented deformable parenchyma, with predominantly bluish colors and some discreet stiff areas (small areas in yellowish color), suggesting acute pancreatic alterations. In only one case (11%), i.e. animal 4, the parenchyma showed to be heterogeneous with reddish areas, demonstrating he stiffness of the tissue, suggesting a chronic alteration (Figure 1). Many studies point that this parenchymal characteristic is related to tissue malignancy, such as in mammary neoplasms, for example [14][15][16], however, these changes were also seen in chronic infl ammatory processes of the pancreatic parenchyma [17,18].
Quantitative elastography presented mean shear wave velocity of 1.91±0.32m/s in GS and 2.35 ±0.53m/s in the GD group. This difference was considered signifi cant (P = 0.014), thus, when pancreatic SWV is higher than 1.98m/s it might indicate pancreatic disease, with a 78% sensitivity, 69% specifi city and 78% AUC (Figure 2). Among GD, the changes caused by acute infl ammatory processes apparently resulted in lower SWV than in chronic alterations.
Goertz et al. [18] cited that, in human patients, chronic infl ammation leads to fi brotic changes of the pancreatic tissue and, in some cases, calcifi cations. These characteristics decrease elasticity of the diseased pancreas, promoting elastographic fi ndings of greater stiffness [19], as observed in the present study. When tissues with sonographic aspects of acute (infl ammatory) cases were observed, they presented discreet areas in yellowish and reddish colors in eight cases. It suggests an increase in parenchyma stiffness in comparison to the healthy group, which presented bluish and greenish on the elastogram (not hard). In one patient with fi ndings compatible with chronicity, a higher stiffness was observed, with bigger and more frequent reddish areas. Although in the present study there was no suspicion of neoplastic lesions, it is suspected that stiffness of these tissues may be higher than in chronic lesions, which might aid in the differential diagnosis of chronic pancreatitis and pancreatic neoplasms, which is still challenging [20]. Further studies are necessary in order to contribute for diagnosis of these alterations.
In both GS and GD, Color Doppler ultrasound did not show neovascularizations and the vascular pattern was homogenous. In Spectral Doppler, mean value of resistive index was 0.74±0.09 in GS and 0.79±0.13 in GD, with no signifi cant difference between these groups (P=0.386). Changes in these parameters were observed in cases of lesions suggestive of malignance in other tissues, such as presence of neovascularization, vessels with a tortuous aspect, elevated systolic and diastolic velocities and changes in the resistive index [21][22][23].
Microbubble contrast-enhanced ultrasonography presented homogenous fi lling of the pancreas. Both groups had no side effects due to the use of the contrast agent. Mean values obtained in the contrast fi lling times did not have signifi cant differences between groups; wash in (P=0.128), peak enhancement (P=0.181) and wash-out (P=0.169) ( Table 2). Although there was no difference between groups, animal 4 (Dalmatian) from the GD presented a markedly prolonged time in the three moments: 12s for the entrance, 32s peak enhancement and 110s the contrast agent was still present in the parenchyma. Studies performed by Rademacher et al. [4] and Lim et al. [9] also presented an increase in the time of entrance and peak enhancement in dogs with pancreatitis.  Results of CEUS technique were not effective in differentiation of altered tissues in most cases, however, it was possible to detect microvascularization, contrary to the study performed by Cai et al. [24] which demonstrated that in cases of acute pancreatitis, this method allows successful identifi cation of changes in comparison to B mode ultrasonography, with an accuracy of 78.0% (103/132) and 47.7% (63/132), respectively. Vanderperren et al. [25] also verifi ed a differentiation of exocrine and endocrine pancreatic tumors using CEUS: adenocarcinomas were presented hypovascular and insulinomas were hypervascular.