Welcome to the all-new Vetlearn

  • Vetlearn is getting a new home. Starting this fall,
    Vetlearn becomes part of the NAVC VetFolio family.

    You'll have access to the entire Compendium and
    Veterinary Technician archives and get to explore
    even more ways to learn and earn CE by becoming
    a VetFolio subscriber. Subscriber benefits:
  • Over 500 hours of interactive CE Videos
  • An engaging new Community for tough cases
    and networking
  • Three years of NAVC Conference Proceedings
  • All-new articles (CE and other topics) for the entire
    healthcare team

To access Vetlearn, you must first sign in or register.

registernow

  • Registration for new subscribers will open in September 2014!
  • Watch for additional exciting news coming soon!
Become a Member

Veterinary Therapeutics Winter 2008 (Vol 9, No 4)

Sensitivity of Serum Markers for Pancreatitis in Dogs with Macroscopic Evidence of Pancreatitis

by Jorg Steiner, MedVet, Dr.med.vet, PhD, DACVIM, DECVIM-CA, Shelley Newman, DVM, DVSc, DACVP, Panagiotis Xenoulis, DVM, Dr.med.vet, Kristen Woosley, DVM, DACVECC, Jan Suchodolski, med.vet, Dr.med.vet, PhD, David Williams, MA, VetMB, PhD, DACVIM, DECVIM-CA, Linda Barton, DVM, DACVECC

    Clinical Relevance

    Pancreatitis is recognized as an important and common problem in dogs, but diagnosis can be challenging. Recently, new assays for the measurement of trypsin-α1-proteinase inhibitor complexes and canine pancreatic lipase immunoreactivity (cPLI and Spec cPL) have been developed and analytically validated. This is the first report of a direct comparison of the sensitivity of these and other more traditional serum markers for the diagnosis of canine pancreatitis in a subset of dogs with this disease (i.e., dogs with both macroscopic and microscopic changes characteristic of pancreatitis). Serum cPLI and Spec cPL concentrations showed the highest sensitivity for the diagnosis of pancreatitis in this group of patients. Further studies will be required to compare the specificity of these serum markers and thus determine their overall clinical utility.

    Introduction

    Pancreatitis is considered to occur commonly in dogs, but only limited data about the true prevalence of this condition in dogs are available. In a single postmortem study of 9,342 dogs, the prevalence of pancreatitis was 1.0%,1 but it has been speculated that this number underestimates the true prevalence of pancreatitis in dogs.2,3 Pancreatitis can be acute or chronic and can vary widely in severity, ranging from subclinical to severe clinical disease. Severe pancreatitis is associated with both local and systemic complications and can lead to significant morbidity and mortality.4 Some cases of canine pancreatitis are associated with classical clinical signs, such as vomiting and abdominal pain, whereas others are associated with various signs, such as anorexia, weakness, diarrhea, fever, or behavioral changes.5 Therefore, the diagnosis of canine pancreatitis can be challenging.

    Abdominal ultrasonography is a valuable tool for the diagnosis of pancreatitis, and a variety of ultrasonographic changes have been reported, including enlargement of the pancreas, fluid accumulation around the pancreas, echogenicity changes within and around the pancreas, a pancreatic mass effect, and a dilated major duodenal papilla.5,6 The highest reported sensitivity of abdominal ultrasonography for canine pancreatitis is 68%.5 However, it should be noted that the patients in that particular study had severe forms of acute pancreatitis (i.e., their pancreatitis was considered to be fatal by the study authors), and empiric clinical data suggest that abdominal ultrasonography would be less sensitive in less severe cases (i.e., those not associated with significant pancreatic necrosis).5

    Serum amylase and lipase activities have traditionally been used for the diagnosis of canine pancreatitis but are neither very specific nor very sensitive for the diagnosis of this disease in dogs.7 The assay for serum canine trypsin-like immunoreactivity (cTLI) measures trypsinogen (and trypsin, if present) and has been shown to be highly sensitive and specific for the diagnosis of exocrine pancreatic insufficiency (EPI), but preliminary evidence suggests that this assay lacks sensitivity for the diagnosis of canine pancreatitis.8,9 Recently, new assays for the measurement of serum concentrations of pancreatic lipase and trypsin-α1-proteinase inhibitor complexes (T-a1-PI) have been developed and validated for use in dogs.10-12 Under physiologic conditions, virtually no trypsin is present in the vascular space, and thus there should be no T-a1-PI in the serum of normal dogs.13 During pancreatitis, trypsinogen is prematurely activated in the pancreas and can leak into the vascular space, where it is scavenged by a1-proteinase inhibitor, leading to the formation of T-a1-PI.13 It has therefore been theorized that T-a1-PI may serve as a serum marker for canine pancreatitis.12 However, the sensitivity of this marker for canine pancreatitis has not been evaluated to date. Canine pancreatic lipase immunoreactivity (cPLI) was shown to be limited to pancreatic acinar cells, suggesting that cPLI is a specific marker for pancreatic acinar cells.14 Furthermore, dogs with EPI had severely decreased (usually undetectable) serum cPLI concentrations, further suggesting that pancreatic lipase originates exclusively from the exocrine pancreas.15 Finally, preliminary reports suggest that serum cPLI concentration may be highly sensitive for the diagnosis of canine pancreatitis.9,16

    The objective of this study was to compare the sensitivity of several serum markers for pancreatitis in a group of dogs with macroscopic evidence of pancreatitis.

    Materials and Methods

    A total of 22 dogs with macroscopic evidence of pancreatitis were included in this study. These dogs were selected from a group of 208 dogs presented for necropsy to the Department of Pathology at the Animal Medical Center within 6 hours after death (of various causes) and were selected because of the presence of macroscopic evidence of pancreatitis. Peripancreatic fat necrosis (Figure 1), pancreatic hemorrhage, presence of pus when the organ was cut, and a dull granular capsular surface were considered macroscopic evidence of pancreatitis. For each dog, the pancreas was removed in toto, the right pancreatic limb was identified with a piece of suture material, and the whole pancreas was submerged in buffered formalin. Serum samples had been collected from all dogs within 24 hours before death and were stored frozen until analysis.

    Serum amylase and lipase activities and serum cTLI concentrations were measured using commercial assays (Modular Analytics P 800 module, Roche Diagnostics, Indianapolis, IN; Diagnostic Chemicals Limited, Oxford, CT; and Canine TLI Radioimmunoassay Kit, Siemens Healthcare Diagnostics, Los Angeles, CA, respectively). Serum cPLI concentration was measured using two assays: a commercial assay (Spec cPL, Idexx Laboratories, Westbrook, ME) and an in-house immunoassay described previously (cPLI).10 Serum T-a1-PI concentrations were also measured using an in-house immunoassay.12

    The histologic pancreatitis activity index (AI) and histologic pancreatitis chronicity index (CI) were calculated for each pancreas as previously described.17 Briefly, each pancreas was sectioned every 2 cm and each section was evaluated by viewing multiple fields separately for neutrophilic infiltration, lymphocytic infiltration, pancreatic necrosis, peripancreatic fat necrosis, pancreatic edema, pancreatic fibrosis, and pancreatic atrophy. Each individual section was assessed for all these parameters, and AI and CI were calculated based on the absence (0) or presence and severity (1, 2, or 3) of the specific lesion in the section as follows:

    • Grade 0: Absence of specific lesion
    • Grade 1: <10% of the section affected
    • Grade 2: 10% to 40% of the section affected
    • Grade 3: >40% of the section affected

    Also, the mean cumulative severity (MCS) scores were assigned based on the number of sections and the severity score for each section (MCS = S score of single sections/number of sections):

    • MCS >0.0 to ≤1.0 = mild
    • MCS >1.0 to ≤2.0 = moderate
    • MCS >2.0 = severe

    The AI and CI were then calculated for each pancreas based on the formulas in the box . The maximum score for both AI and CI was 3.0. Scores were interpreted as follows:

    • AI >0.0 to ≤1.0 = mild
    • AI >1.0 to ≤2.0 = moderate
    • AI >2.0 = severe
    • CI >0.0 to ≤1.0 = evidence of mild chronicity
    • CI >1.0 to ≤2.0 = evidence of moderate chronicity
    • CI >2.0 = evidence of severe chronicity

    After calculation of AI scores for all dogs, serum parameters were evaluated for correlation with the AI scores.

    The medical records of all patients were evaluated for clinical signs associated with pancreatitis and abdominal ultrasonography reports, when available.

    For each serum parameter tested, the number of dogs with values above the upper limit of the reference range and, if available, a suggested cut-off value for pancreatitis were counted and sensitivities were calculated. Data were tested for normal distribution using a D'Agostino-Pearson omnibus normality test (Prism5, GraphPad, San Diego, CA). Also, the mean AI was compared between dogs that underwent abdominal ultrasonography and those that did not, using a statistical software program (Prism5). Finally, the median AI was compared among the nine dogs that underwent abdominal ultrasonography and between dogs with and without ultrasonographic evidence of pancreatitis using the same statistical software program.

    Results

    A total of 22 dogs with macroscopic evidence of pancreatitis (Table 1) were included in the study. All 22 dogs also had histologic evidence of pancreatic inflammation, but pancreatitis activity scores were low for all dogs (Table 1). None of the dogs had an AI >2.0 and only 4 of 22 dogs (18.2%) had an AI >1.0. Serum amylase and lipase activities were above the upper limit of the reference range in 9 (40.9%) and 7 (31.8%) dogs, respectively (Table 2). Serum amylase and lipase activities were above a suggested cut-off value for pancreatitis (three times the upper limit of the respective reference range) in 4 (18.2%) and 3 (13.6%) dogs, respectively. Serum cTLI concentration was above the upper limit of the reference range (35.0 µg/L) in 8 (36.4%) dogs (Table 2). Serum cPLI concentration as measured by in-house ELISA was above the upper limit of the reference range (102.1 µg/L) in 17 (77.3%) dogs and above a suggested cut-off value for pancreatitis (200 µg/L) in 14 (63.6%) dogs (Table 2). Serum cPLI concentration as measured by Spec cPL was above the upper limit of the reference range (200 µg/L) in 16 (72.7%) dogs and above a suggested cut-off value for pancreatitis (400 µg/L) in 14 dogs (63.6%; Table 2). Finally, serum T-a1-PI concentration was above the upper limit of the reference range (32 µg/L) in 7 (31.8%) dogs (Table 2). There was no correlation between AI and serum amylase activity, AI and serum cTLI concentration, or AI and serum T-a1-PI concentration. There was significant correlation between AI and serum lipase activity (Spearman r = 0.493; P = .0197; Figure 2), AI and serum cPLI concentration as measured by in-house ELISA (Spearman r = 0.530; P = .0111; Figure 3), and AI and Spec cPL (Spearman r = 0.547; P = .0084; Figure 4).

    Clinical signs compatible with pancreatitis were noted in 20 of the 22 study dogs (Table 3): vomiting in 18 of 22 (81.8%) dogs, abdominal pain in 10 of 22 (45.5%), anorexia in 13 of 22 (59.1%), and diarrhea in 8 of 22 (36.4%). Abdominal ultrasonography was performed in 9 of 22 dogs, and evidence of pancreatitis was considered to be present in 6 of these dogs (Table 3). The mean AI was higher for dogs that underwent ultrasonography (0.82) than for dogs that did not (0.47), but this difference was not statistically significant (P = .097). The median AI was significantly higher in dogs that had ultrasonographic evidence of pancreatitis (1.05) than in dogs that did not (0.3; P = .037).

    Discussion

    This study included only dogs with macroscopic evidence of pancreatitis. This was done because of recent evidence suggesting that microscopic findings of pancreatic inflammation are extremely common in dogs, and concerns have been voiced that, in some cases, microscopic disease may not be clinically significant.2 Therefore, we included only dogs that had macroscopic changes, assuming that such dogs would have more severe and thus more clinically relevant disease.2 All of the dogs also had microscopic changes suggestive of pancreatitis and 20 of the 22 dogs with macroscopic evidence of pancreatitis had compatible clinical signs that might have been associated with pancreatitis; however, from what could be gathered from the medical records, pancreatitis was not suspected clinically in many of these dogs, although the differential diagnosis "pancreatitis" may not have been recorded in each record. This suggests that clinically, as has been reported for human patients, pancreatitis is undersuspected and underdiagnosed in dogs.18

    As shown in Table 1 , AI scores were low for most dogs enrolled into this study; the mean AI score was 0.61 (SD ±0.49), no dogs had an AI score >2.0, and only four dogs had an AI score >1.0. This would suggest that dogs enrolled in this study had histologically mild to moderate pancreatitis based on a recently published scoring system.17 It is possible that the sensitivities of all the diagnostic markers evaluated in the dogs in this study would be higher in dogs with histologically more severe pancreatitis. Further studies are needed to evaluate sensitivities in such dogs.

    Serum lipase activity had the lowest sensitivity for macroscopic pancreatitis (13.6%), followed by serum amylase activity (18.2%), serum T-a1-PI concentration (31.8%), and serum cTLI concentration (36.4%). Serum cPLI concentration, as measured by in-house ELISA or Spec cPL, had the highest sensitivities at 63.6% each and also showed a positive correlation with the AI. Although this number may appear low, it should be noted that 81.8% of the dogs enrolled in this study had mild pancreatitis, 18.2% had moderate pancreatitis, and none had severe pancreatitis as judged by histologic AI scores. The weak correlation of serum lipase activity, serum cPLI concentration, and serum Spec cPL concentration would suggest that, at least for these three parameters, sensitivities would be expected to be higher in dogs with more severe disease. This would explain the difference in the sensitivity found in this study when compared with the preliminary data reported in two research abstracts that suggested a sensitivity of serum cPLI concentration for canine pancreatitis of approximately 82%.9,16

    Only serum lipase activity, serum cPLI concentration, and serum Spec cPL concentration showed a positive correlation with AI scores. However, Spearman r values for all three diagnostic parameters were rather low (0.493, 0.530, and 0.547, respectively), suggesting only a very weak correlation. While serum lipase activity, serum cPLI concentration, and serum Spec cPL concentration would generally be expected to be higher in dogs with more severe histopathologic disease, the correlation for all three parameters would be too weak to allow prediction of the histopathologic severity from the measurement of any of these three parameters. These findings agree with empiric data that would suggest that a single measurement of serum cPLI or Spec cPL concentration cannot accurately predict disease severity in dogs with pancreatitis (unpublished personal observations, JMS).

    Unfortunately, abdominal ultrasonography had been performed in only nine of the dogs enrolled in this study. Thus, the number of dogs evaluated was too small to calculate a reliable sensitivity of abdominal ultrasound for pancreatitis. Ultrasonograms of six of the nine dogs examined were interpreted as showing evidence of pancreatitis, which would suggest a sensitivity of 66.7%, which is similar to that for serum cPLI and serum Spec cPL concentrations in this study. It should be noted, however, that the mean AI was higher for the dogs in which abdominal ultrasonography was performed (mean AI, 0.82) than for dogs in which it was not (mean AI, 0.47), although this difference did not reach statistical significance (P = .097). This may suggest that when dogs had more severe disease, clinicians had a higher degree of clinical suspicion for pancreatitis and were more likely to perform or request abdominal ultrasonography; however, this speculation needs to be further verified. Alternatively, clinicians may have suspected other gastrointestinal, hepatic, or abdominal disorders that spurred their decision to request abdominal ultrasonography. Regardless, additional studies are needed to compare the sensitivity of abdominal ultrasonography with that of serum markers for pancreatitis. Also, the dogs that had ultrasonographic evidence of pancreatitis were those with the highest AI, and the median AI score was significantly higher for dogs that had ultrasonographic evidence of pancreatitis than those that did not (1.05 vs. 0.3; P = .037; Figure 5). This would suggest that, especially in cases with more severe pancreatic pathology, abdominal ultrasonography can serve as a reliable diagnostic tool for pancreatitis. Two of the three dogs that had no evidence of pancreatitis on abdominal ultrasound had serum cPLI concentrations above the reference range and above the cut-off value for pancreatitis as measured by both the in-house ELISA for cPLI and the Spec cPL.

    One of the most common grossly apparent manifestations of macroscopic pancreatitis in this study was peripancreatic fat necrosis revealed by the presence of saponified fat as shown in Figure 1 . No documentation of the duration of the persistence of saponified fat after an episode of pancreatitis could be found in the literature. Thus, there is a possibility that saponified nodules may have formed in these dogs as a result of a previous episode of pancreatitis, and that at the time of necropsy the dogs did not have clinically significant active pancreatitis. However, all 22 dogs had histopathologic evidence of pancreatic inflammation, 19 dogs showed neutrophilic pancreatic infiltration, 16 showed evidence of pancreatic necrosis, and 18 showed evidence of peripancreatic fat necrosis. Therefore, it appears likely that all dogs had active pancreatic inflammation at the time of necropsy.

    Conclusion

    In this group of dogs with macroscopic pancreatitis, serum cPLI concentration as measured by an in-house ELISA and by a commercial assay (Spec cPL) was the most sensitive serum marker for pancreatitis. In the limited number of dogs in which it was performed, abdominal ultrasonography also appeared to be useful in diagnosing pancreatitis and may be more sensitive in dogs with more severe pancreatic pathology. These findings would suggest that, together with all other available historical and clinical data, the combination of abdominal ultrasonography performed by an experienced operator and serum cPLI or Spec cPL concentration would yield the highest sensitivity for diagnosing canine pancreatitis. Further studies are needed to compare the specificities of these different serum markers and thus to be able to compare their overall diagnostic utility.

    Downloadable PDF

    A portion of this material was presented at the 25th Annual Forum of the American College of Veterinary Internal Medicine in Seattle, WA, June 2007.

    Reprint requests should be directed to Dr. Steiner: phone, 979-862-4046; e-mail, jsteiner@cvm.tamu.edu.

    1. Hänichen T, Minkus G. Retrospektive Studie zur Pathologie der Erkrankungen des exokrinen Pankreas bei Hund und Katze. Tierärztliche Umschau 1990;45: 363-368.

    2. Newman S, Steiner J, Woosley K, et al. Localization of pancreatic inflammation and necrosis in dogs. J Vet Intern Med 2004;18:488-493.

    3. Steiner JM. Is it pancreatitis? Vet Med 2006;101:158-167.

    4. Bradley EL. A clinically based classification system for acute pancreatitis. Arch Surg 1993;128:586-590.

    5. Hess RS, Saunders HM, Van Winkle TJ, et al. Clinical, clinicopathologic, radiographic, and ultrasonographic abnormalities in dogs with fatal acute pancreatitis: 70 cases (1986-1995). JAVMA 1998;213:665-670.

    6. Saunders HM. Ultrasonography of the pancreas. Probl Vet Med 1991;3:583-603.

    7. Strombeck DR, Farver T, Kaneko JJ. Serum amylase and lipase activities in the diagnosis of pancreatitis in dogs. Am J Vet Res 1981;42:1966-1970.

    8. Williams DA, Batt RM. Sensitivity and specificity of radioimmunoassay of serum trypsin-like immunoreactivity for the diagnosis of canine exocrine pancreatic insufficiency. JAVMA 1988;192:195-201.

    9. Steiner JM, Broussard J, Mansfield CS, et al. Serum canine pancreatic lipase immunoreactivity (cPLI) concentrations in dogs with spontaneous pancreatitis [abstract]. J Vet Intern Med 2001;5:274.

    10. Steiner JM, Teague SR, Williams DA. Development and analytic validation of an enzyme-linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in serum. Can J Vet Res 2003;67:175-182.

    11. Steiner JM, Williams DA. Development and validation of a radioimmunoassay for the measurement of canine pancreatic lipase immunoreactivity in serum of dogs. Am J Vet Res 2003;64:1237-1241.

    12. Suchodolski JS, Collard JC, Steiner JM, et al. Development and validation of an enzyme-linked immunosorbent assay for measurement of a1-proteinase inhibitor/trypsin complexes in canine sera [abstract]. J Vet Intern Med 2001;15:311.

    13. Nakae Y, Naruse S, Kitagawa M, et al. Activation of trypsinogen in experimental models of acute pancreatitis in rats. Pancreas 1995;10:306-313.

    14. Steiner JM, Berridge BR, Wojcieszyn J, et al. Cellular immunolocalization of gastric and pancreatic lipase in various tissues obtained from dogs. Am J Vet Res 2002;63:722-727.

    15. Steiner JM, Rutz GM, Williams DA. Serum lipase activities and pancreatic lipase immunoreactivity concentrations in dogs with exocrine pancreatic insufficiency. Am J Vet Res 2006;67:84-87.

    16. Sinclair HM, Fleeman LM, Rand JS, et al. Continuing pancreatic inflammation or reduced exocrine function are common in dogs after acute pancreatitis [abstract]. J Vet Intern Med 2006;20:750.

    17. Newman SJ, Steiner JM, Woosley K, et al. Histologic assessment and grading of the exocrine pancreas in the dog. J Vet Diagn Invest 2006;18:115-118.

    18. Lowenfels AB. Epidemiology of diseases of the pancreas: clues to understanding and preventing pancreatic disease. In Grendell JH, Forsmark CE, eds. Controversies and Clinical Challenges in Pancreatic Diseases. Bethesda. Md: American Gastroenterological Society, 1998;9-13.

    References »

    NEXT: Comparative Efficacy of Tilmicosin versus Tulathromycin as a Metaphylactic Antimicrobial in Feedlot Calves at Moderate Risk for Respiratory Disease

    didyouknow

    Did you know... In feline pemphigus foliaceus, the face, ears, and feet are most commonly involved. Read More

    These Care Guides are written to help your clients understand common conditions. They are formatted to print and give to your clients for their information.

    Stay on top of all our latest content — sign up for the Vetlearn newsletters.
    • More
    Subscribe