• Open Access

Feasibility and Safety of Endoscopic Ultrasound-Guided Fine Needle Aspiration of the Pancreas in Dogs

Authors


Corresponding author: Dr med. vet. P.H. Kook, Dipl. ACVIM & ECVIM-CA, Clinic for Small Animal Internal Medicine, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland; e-mail: peterhendrikkook@gmail.com

Abstract

Background

Endoscopic ultrasound (EUS)-guided fine needle aspiration (EUS-FNA) has proven a useful and safe diagnostic tool for assessing pancreatic disease in human medicine. No information about pancreatic EUS-FNA is available in dogs.

Objectives

To assess the feasibility and safety of pancreatic EUS-FNA in healthy dogs.

Animals

Thirteen beagles with a median body weight of 13.4 kg.

Methods

Experimental study. An ultrasound endoscope (insertion tube outer diameter 11.8 mm) was used, and FNA was carried out with 19 G needles. The optimal puncture site was chosen with the aid of Doppler imaging. Complete clinicopathologic assessments including pain scoring and pancreas-specific lipase measurements were obtained before EUS as well as on day 1 and day 2 after EUS-FNA.

Results

The pancreatic body was identified in all dogs, the left lobe was clearly identified in 9/13 and appeared indistinctly marginated in 4/13 dogs, and the distal third of the right lobe could not be identified in 7/13 dogs. EUS-FNA was carried out in 12/13 dogs. Cellularity of smears was adequate for evaluation in 8/12 cases, in which samples were obtained transgastrically (n = 4) or transduodenally (n = 4). All dogs recovered uneventfully and no clinical and laboratory abnormalities occurred during the 48 hour monitoring period after the procedure.

Conclusion and Clinical Importance

Although the healthy canine pancreas is difficult to visualize in its entirety with EUS, pancreatic EUS-FNA with a 19 G needle is feasible in medium-sized dogs and can be considered a safe procedure. Its diagnostic usefulness should be evaluated in dogs with pancreatic disease.

Abbreviation
EUS

endoscopic ultrasound

Endoscopic ultrasound (EUS) involves the use of an endoscope fitted with an ultrasound probe. The mucosa and lumen of hollow organs are visualized with the endoscope whereas the ultrasound probe permits imaging of tissues below the mucosa as well as adjacent structures. The closer proximity of the ultrasound probe to deep abdominal structures being imaged facilitates the use of high frequencies.Furthermore, depending on the size of the patient, and the presence of luminal gas in the gastrointestinal tract, deeper structures such as the pancreas can sometimes be difficult to assess with transabdominal ultrasound (AUS). Therefore EUS can be superior in identifying such structures and can provide images with a higher resolution. EUS was first introduced in the early 1990s and has since become a cornerstone technique for assessing abnormalities in the abdominal cavity and retroperitoneal space in humans.[1] This procedure allows optimal visualization as well as ultrasound-guided fine needle aspiration (FNA) of tissues.[1] EUS has not been used widely in veterinary medicine. Video ultrasound gastroscopy was used to examine the canine pancreas in experimental dogs,[2] as well as in the work-up of intrathoracic lesions in 2 dogs.[3] A standardized procedure for examination of the canine abdomen, including a detailed EUS description of the pancreas with anatomical landmarks, has been described recently.[4] Another recent publication compared AUS and EUS for diagnosis of feline pancreatitis and concluded that EUS may be superior for imaging the pancreas, particularly in obese cats.[5] However, no attempt was made to obtain EUS-guided fine needle aspiration (EUS-FNA) samples of the pancreas for cytologic evaluation in that study. Although a presumptive diagnosis of acute pancreatitis can usually be made based on clinical signs, laboratory results, and AUS findings, cytological or histopathologic examination of pancreatic tissue is required for the diagnosis of chronic inflammatory, cystic, or neoplastic diseases. In people, EUS-FNA of the pancreas with a curved linear-array ultrasound endoscope is considered a safe procedure.[6] Major risks reported with EUS-FNA are infection, intra- and extraluminal bleeding, and acute pancreatitis.[7] To the authors' knowledge, EUS-FNA of the pancreas has not been performed in dogs. Because EUS-FNA would eliminate the need for more invasive procedures such as laparoscopy or laparotomy, the goal of the present study was to assess the feasibility and safety of EUS-FNA of the pancreas in dogs. The hypothesis of this study was that EUS-FNA of the pancreas is a safe procedure and does not result in clinicopathologically detectable postinterventional complications.

Materials and Methods

This study was approved by the Committee for the Permission of Animal Experimentation, Canton of Zurich, Zurich, Switzerland.

Study Design

Dogs

Thirteen Beagles ranging in age from 1 to 5 years (median 2 years) and weighing 10.9‒15.7 (median 13.4 kg) kg were used. There were 8 intact females and 6 intact males. The dogs were considered healthy based on the history and the results of physical examination, complete blood cell count (CBC), serum biochemistry profile (SBP), and urinalysis. The dogs were fasted for 16 hours before EUS. A blood sample for a CBC, SBP including amylase and lipase activities, and canine pancreas-specific lipase (Spec cPL) was collected during placement of an indwelling catheter for anesthesia (day 0). The same variables were measured again on days 1 and 2 in the morning. The serum samples for Spec cPL measurements were stored at −20°C and when all collections had been completed, they were shipped on dry ice for analysis. All samples were measured on a single microtiter plate, and the assay was carried out according to the manufacturer's directions.[8] The reportable dynamic range of the assay was 30–1000 μg/L, and Spec cPL results <200 μg/L were considered normal.

Examination Procedure

The EUS examinations were done with an ultrasound videogastroscope1 (operating channel 3.7 mm, insertion tube outer diameter 11.8 mm) with an Albarran elevator to facilitate needle positioning, which was supported by an ultrasound unit.2 This ultrasound endoscope used a curved linear array transducer with a scanning range of 0‒180° parallel to its long axis. The probe has a fundamental frequency range of 5 to 10 MHZ. The dogs were sedated with acepromazine3 (0.03 mg/kg) and buprenorphine4 (0.014 mg/kg). General anesthesia was induced with propofol5 and maintained with isoflurane6 (1.1–1.3% in 100% oxygen). All ultrasonographic images were stored on the built-in hard disk drive of the ultrasound machine and simultaneously sent to the picture archiving and communication server of the clinic for further evaluation.

EUS Examination Technique and EUS-Guided FNA

With the dog in left lateral recumbency, the ultrasound endoscope was inserted with the transducer facing down (toward the dog's left side) in the straight or neutral position, and advanced into the gastric cardia until the liver was visualized. From this point the cranial abdominal region was examined by turning the endoscope clockwise and counterclockwise, each maneuver combined with up, down, left, and right turns of the dials to visualize each organ. Readily identifiable landmarks that were used were the portal vein and caudal vena cava, spleen, cranial duodenal flexure, left kidney, and aorta as described previously.[2] The liver, gallbladder, bile ducts, kidneys, adrenal glands, and body and left limb of the pancreas were scanned from the stomach. The ultrasound endoscope was then advanced through the pylorus into the duodenum and the right limb of the pancreas was identified. After completion of the EUS examination, the optimal site for FNA of the pancreas was identified by power Doppler imaging to avoid accidental puncture of interposed vessels such as the Vena pancreaticoduodenale. Aspiration was carried out with a 19 ‒ gauge (G) EUS-needle.7 One single-use disposable needle was sterilized and reused in 1 dog; new needles were used in all other cases. The EUS needle was manipulated by a piston integrated into the biopsy handle. The needle was supported by a stable metal spiral sheath, which was firmly connected to the handle. After the transducer was positioned securely in front of the targeted pancreatic tissue, the metal spiral was inserted and the handle with the Luer-lock firmly screwed onto the biopsy channel. After carefully confirming the correct transducer position, the needle with the attached stylet was advanced under EUS-guidance to determine its direction. The distance between the projecting distal end of the needle sheath and the puncture target was measured to avoid overshooting the puncture target. The handle had 1-cm interval marks and an adjustable “needle stopper”; setting the stopper at the appropriate interval on the handle prevented the needle from being advanced beyond the desired depth of insertion. With the stylet retracted a few millimeters, the biopsy needle was moved forward into the pancreatic tissue with a quick strong thrust of the handle. With the tip of the needle positioned approximately in the middle of the target, the stylet was completely removed and a 10-mL syringe with luer stopcock was attached and used to apply negative pressure while the needle was moved to and fro inside the pancreas under ultrasonographic control. With the needle tip still in the pancreas, the negative pressure was slowly released and the needle retracted into the needle sheath and locked in that secure position. After removing the needle assembly, the aspirate was immediately expelled onto specimen slides. The samples were allowed to air-dry for subsequent cytologic assessment. All dogs were reevaluated ultrasonographically (AUS) 20 to 30 minutes post-EUS-FNA and checked for evidence of free fluid. Postprocedural analgesia consisted of one-time administration of metamizole8 (30 mg/kg BW, IM). Post-EUS demeanour was assessed with a standardized pain scoring (PS) system for measurement of postoperative pain in dogs.[9] Assessed criteria included physiologic data (heart and respiratory rates), response to palpation, activity, mental status, posture, salivation and vocalization. The minimum possible total pain score generated with this scale is 0 and the maximum possible score is 27. All dogs were examined 2 to 3 hours after the procedure (t0) and again in the morning and evening of the following 2 days.

Cytologic Examination

The cytologic smears were air-dried and stained (modified Wright stain) on the same day with an autostainer.8 Cytologic assessment included evaluation of the quality of the smear based on thickness, cellularity, morphology of the cells, and background material.

Results

EUS-FNA

The median total EUS examination time including the EUS-guided FNA was 112 minutes (range, 105–138); the actual EUS-FNA procedure of the pancreas required a median of 14 minutes (range, 8–23). By means of the pylorus and cranial duodenal flexure as landmarks, the body of the pancreas could be identified in all 13 dogs. The distal third of the right limb of the pancreas, where it becomes physically separated from the duodenum, was difficult to visualize and could only be identified in 6/13 dogs (Fig 1). In those cases the V. pancreaticoduodenale served as an important landmark. The left pancreatic limb was difficult to visualize. It could be clearly identified in 9/13 dogs, and was indistinctly marginated in 4/13 dogs. Visualization of the distal third of the left limb of the pancreas was difficult as this part of the pancreas courses away from the stomach and its position varies. The pancreas appeared isoechoic (9/13) or hypoechoic (4/13) relative to the surrounding mesentery. Although the pancreatic borders were not distinct in all cases, identification of the organ was facilitated by its more homogeneous appearance compared with the surrounding mesentery as well as by visualization of the pancreatic duct or its location being attached to the duodenum (Fig 2). The cranial pancreaticoduodenal artery was seen branching from the gastroduodenal artery within the right lobe of the pancreas in 8/13 dogs. The mean thickness of the left pancreatic lobe was 0.82 ± 0.18 cm and the mean thickness of the right pancreatic lobe was 0.91 ± 0.19 cm.

Figure 1.

The endosonoscope is placed in the stomach and the image is directed to the region of the pylorus. The pancreas is outlined with small white arrows. Note the homogeneous echo texture of the pancreas landmarked by the Vena pancreatic duodenale. The duodenum is partly visible as the free part of the pancreas is shown.

Figure 2.

The proximal part of the right pancreatic limb is shown with the endoscope placed in the stomach right at its proximal part next to the pylorus. The duodenum is filled with ingesta. The pancreas is outlined with small white arrows. The echo texture of the pancreas is very homogeneous and well delineated.

The ideal pancreatic EUS-FNA site varied among dogs. The final determination where to aspirate from was decided individually based on ideal image quality as well as ideal visualization of the target organ. The pancreas was aspirated transgastrically in 6/13 dogs, and a transduodenal approach was chosen in 7/13 dogs (Fig 3). In 1 dog, in which a resterilized needle was used, transmural penetration was insufficient, and EUS-FNA was not possible. While continuously aspiratng, at least 6-8 rapid intrapancreatic needle passes were made in all examinations.

Figure 3.

The endoscope is inserted into the duodenum from where it is directed medially to visualize the pancreas, that is outlined with small white arrows. In this case, the pancreas is less homogeneous, but still well delineated. It is landmarked by the Vena pancreatic duodenale.

Postprocedure ultrasonographic examination showed scant amounts of free peripancreatic fluid in 1 of the 12 dogs, in which pancreatic EUS-FNA was successful. All dogs recovered uneventfully and no further analgesia was required based on clinical examination and results of pain scores. The postoperative pain score was minimal in all dogs (median 0, range 0–4) at t0, and 0 at all following time points.

Cytology

A mean of 8 FNA smears were assessed subjectively in each dog. Cellular yield varied greatly, and there was evidence of normal exocrine pancreatic cells in 8 of 12 cases. In 5 of 8 cases, pancreatic cells were found in only 1 slide per animal. The pancreatic cells had slightly granular eosinophilic cytoplasm and a round to oval nucleus with fine cribriform chromatin and a prominent nucleolus. The cells were embedded in a moderately basophilic proteinaceous background. In 4 dogs, exocrine pancreatic cells were limited to very small clusters randomly distributed among several duodenal villus epithelial cells or gastric or squamous epithelial cells, accompanied by contaminants from the oral cavity. Overall, the cytologic yield did not correlate with the aspiration route (transgastric versus transduodenal). A mixed population of bacteria and many partially digested skeletal muscle fibers were visible in the background in all 4 aspirates collected transduodenally, but in only 1 aspirate obtained transgastrically. In 1 transduodenally obtained sample, 1 smear had only exocrine pancreatic epithelial cells arranged in small conglomerates around very fine blood vessels (Fig 4).

Figure 4.

Pancreatic EUS-FNA cytology, modified Wright stain: Numerous polyedric cells with abundant bluish cytoplasm and numerous eosinophilic, 1″ in diameter, round, intracytoplasmic zymogen granules. The nuclei are round to oval with dense chromatin and 1–2 round nucleoli. Next to the exocrine pancreatic cells are several capillaries (arrow) with intraluminal erythrocytes (star) visible.

Laboratory Assessment

All hematological and biochemical values were within reference ranges at all time points. Serum amylase and lipase activities did not change significantly over time. The cPL concentration was well below 200 μg/L in all serum samples at all time points. The concentrations were only measurable in 7 samples and ranged from 31 to 134 μg/L.

Discussion

This study demonstrates the feasibility and safety of EUS-FNA of the pancreas in medium-sized dogs. The results of repeated clinical examinations and biochemical analyses over 2 days indicated that EUS-FNA of the pancreas with a 19 G needle is minimally invasive. The pain score results clearly indicated that aspirations with a 10 mL syringe for suction did not lead to clinically apparent pancreatitis. This is in agreement with the results of EUS-guided pancreatic biopsy in people. In a recent multicenter survey evaluating complications after this procedure at EUS centers, iatrogenic acute pancreatitis had occurred in 9/13,223 (0.07%) cases.[7] A higher incidence of complications (2%) was reported in a prospective study,[10] and it is generally believed that the overall risk of pancreatitis is higher when small pancreatic lesions are aspirated because the potential for pancreatic ductal tissue damage increases.[11] In this context, the results of the present study are of particular interest because to our knowledge, EUS-FNA of healthy and therefore small pancreatic tissue diameters has never been reported previously. A recent study in dogs showed that AUS-guided pancreatic FNA with a 22 G needle did not cause significant damage to the pancreas based on Spec cPL activity, which remained within the reference range.[12] In that study, the activity of Spec cPL was measured immediately after pancreatic FNA. This study monitored Spec cPL activities over the course of 48 hours and based on the normal values it was concluded that intermediate-term adverse effects of pancreatic tissue sampling are unlikely.

In humans, EUS-FNA for pancreatic disease is usually performed with a 22 G needle. We have chosen a large caliber needle such because a 19 G needle for the purpose of the present study, because the relatively small amount of tissue samples obtained from a 22 G needle may limit its diagnostic utility in pancreatic disease.

Iatrogenic infection is another complication reported in humans undergoing EUS-guided FNA.[13] Although constant firm contact between the transducer and the gastrointestinal wall near the target pancreatic tissue was attempted, the stomach or intestinal wall may have been punctured several times because cytologic smears often contained bacteria. However, clinical and hematological signs of infection were not seen in any of the dogs; in addition, problems were not encountered by the caretaker after the dogs had been returned to their kennels.

It was surprising that visualization of the entire pancreas was difficult even though state-of-the-art EUS equipment with a high resolution was used. The body of the pancreas, which is the largest part, was the easiest to identify and was seen in all dogs. The fact that it was located within an easily identifiable region consisting of the proximal duodenum, the pylorus, and the adjacent stomach wall where its position was most consistent contributed to ease of identification. The proximal portions of the left and right limbs of the pancreas were also easily identified, but identification of the parts of the pancreas that were not adjacent to the duodenum or stomach was more difficult. These problems were not encountered in a recent description of the EUS technique in 4 dogs, where especially the left pancreas seemed comparatively easy to be visualized.[4] This discrepancy may be because of differences in user experience; likewise chances to miss parts of the pancreas may also increase with the larger number of dogs examined in the present study. This problem was exacerbated by the fact that the echogenicity of the pancreatic parenchyma was similar to that of the surrounding mesentery. We felt that the transgastric approach provided more flexibility than the duodenal approach because it allowed different views of the pancreas. A major problem of the duodenal approach was that the duodenum and attached pancreas moved simultaneously when the scope was repositioned, which made visualization of the pancreas difficult.

Visualization of the pancreas for FNA was also hampered by the fact that it was difficult to keep the sonographic image of the aspiration target in place during manipulation of the biopsy device. For example, when the ultrasound endoscope was bent maximally at 180°, there was considerable strain on the sheath, biopsy needle, and stylet, necessitating straightening and readjustment of the scope. This had a negative effect on transducer position and angle of the needle and made FNA difficult.

The presence of a pathologist during the endoscopy procedure has been recommended for confirmation of sufficient sample collection.[14] In our study, there was no rapid on-site cytologic evaluation, which may be considered an inadequacy of the study design. On-site evaluation means that FNA can be repeated to ensure that adequate material for cytologic evaluation has been collected before the patient is recovered from anesthesia. It has also been postulated that a key to adequate specimen cellularity is to avoid using the same aspiration route more than once. This is accomplished by means of the endoscope forceps elevator (the Albarran elevator) at the time of aspiration, which ensures that the needle takes different routes in the target tissue.[15] However, this approach was not feasible in our study because the target was small and there were no pancreatic masses.

Another likely reason why pancreatic cells were not recovered in 4 of 12 dogs might be the tight fibrovascular network of the healthy canine pancreas preventing cells from exfoliating readily. This problem was recently described in a study with a AUS-guided 22G 3-inch spinal needle in dogs.[12]

In addition, the skill and experience of the operator play a major role in the diagnostic yield of cells in EUS-FNA. The author who performed EUS-FNA in this study (PK) had practiced on cadavers and anatomically shaped plastic phantoms before starting this experiment, but had not done the procedure in live animals. In human medicine, the percentage of nondiagnostic specimens from EUS-FNA in a gastroenterological tertiary referral center decreased significantly after a training period. Learning curves for EUS-FNA depended on the target tissues and were steeper for lymph nodes than for pancreatic lesions.[16] The recorded times (EUS examination and EUS-FNA procedure) in the present study did not change over the course of the project, yet all procedures during this experiment were undertaken in healthy animals under ideal anesthetic conditions, without the pressure to account the work performed. Although it must be acknowledged that prolonged procedures requiring anesthesia and operator time is going to be expensive and may carry some significant risk in animals with pancreatic disease, EUS-FNA times will probably be much shorter in more circumscribed lesions.

In summary, our study confirmed that EUS-FNA of the pancreas is feasible and safe in medium-sized dogs and that complete visualization of the entire healthy pancreas is difficult. Further studies in dogs with pancreatic disease are needed to assess the clinical usefulness of this procedure.

Acknowledgments

We thank Mrs Esther Merz for her great care and assistance with the Beagles.

Footnotes

  1. 1

    Olympus GF-UC140P-echoendoscope, Olympus, Hamburg, Germany

  2. 2

    Aloka Prosound SSD alpha 10

  3. 3

    Prequilan, Fatro, SPA, Ozzano Emilia, Italy

  4. 4

    Temgesic, Essex Pharma GmbH, Germany

  5. 5

    Propoflo, Abbot AG, Baar, Switzerland

  6. 6

    IsoFlo

  7. 7

    EchoTip-Ultra, Cook, Switzerland AG Medical Products Vetalgin N ad us. vet. Veterinaria AG, Pfäffikon, Switzerland Hematek, Siemes, Zürich, Switzerland

  8. 8

    Hematek Siemens

Ancillary