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Keywords:

  • Amylase;
  • Lipase;
  • Pancreas-specific lipase;
  • Pancreatic inflammation;
  • Trypsinogen-like immunoreactivity

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background

Pancreatitis is a common disorder in dogs for which the antemortem diagnosis remains challenging.

Objectives

To compare the sensitivity and specificity of serum markers for pancreatitis in dogs with histopathologic evidence of pancreatitis or lack thereof.

Animals

Seventy dogs necropsied for a variety of reasons in which the pancreas was removed within 4 hours of euthanasia and serological markers were evaluated within 24 hours of death.

Methods

Prospective study: Serum was analyzed for amylase and lipase activities, and concentrations of canine trypsin-like immunoreactivity (cTLI) and canine pancreas-specific lipase (cPL). Serial transverse sections of the pancreas were made every 2 cm throughout the entire pancreas and reviewed using a semiquantitative histopathologic grading scheme.

Results

The sensitivity for the Spec cPL (cutoff value 400 μg/L) was 21 and 71% in dogs with mild (n = 56) or moderate-severe pancreatitis (n = 7), and 43 and 71% (cutoff value 200 μg/L), respectively. The sensitivity for the cTLI, serum amylase, and lipase in dogs with mild or moderate-severe pancreatitis was 30 and 29%; 7 and 14%; and 54 and 71%, respectively. The specificity for the Spec cPL based on 7 normal pancreata was 100 and 86% (cutoff value 400 and 200 μg/L, respectively), whereas the specificity for the cTLI, serum amylase, and lipase activity was 100, 100, and 43%, respectively.

Conclusion and Clinical Importance

The Spec cPL demonstrated the best overall performance characteristics (sensitivity and specificity) compared to other serum markers for diagnosing histopathologic lesions of pancreatitis in dogs.

Abbreviations
CI

confidence interval

cPL

canine pancreatic lipase

cTLI

canine trypsin-like immunoreactivity

ELISA

enzyme-linked immunosorbent assay

HE

hematoxylin & eosin

RIA

radioimmunoassay

WSAVA

World Small Animal Veterinary Association

Although pancreatitis is a relatively common disorder in dogs, the antemortem diagnosis of this disease remains clinically challenging. Clinical signs are protean and nonspecific, and can include anorexia, vomiting, lethargy, diarrhea, melena, weight loss, hematemesis, and hematochezia.[1] Risk factors for acute pancreatitis in dogs include obesity, diabetes mellitus, hyperadrenocortcisim, hypothyroidism, prior gastrointestinal disease, and epilepsy, and Miniature Schnauzers and Miniature Poodles appear overrepresented.[2]

Many diagnostic procedures and tests have been evaluated for their sensitivity and specificity for pancreatic disease detection. Measurement of serum lipase and amylase activity has been shown to lack specificity following the demonstration that pancreatectomy does not significantly alter their activity.[3] In addition, increases in serum lipase activity occur in dogs with renal and hepatic disease in the absence of pancreatic inflammation.[4, 5] Measurement of serum trypsin and trypsinogen-like immunoreactivity (TLI) is highly sensitive and specific for the diagnosis of exocrine pancreatic insufficiency; however, this assay has limited value for the diagnosis of canine pancreatitis.[6, 7] More recently, a radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were developed and validated for the measurement of canine pancreatic lipase (cPL).[8, 9] The canine pancreatic lipase immunoreactivity (cPLI) assay has immunoreactivity specific for pancreatic acinar cells.[10] The cPLI assay has been refined by using monoclonal antibodies in a sandwich ELISA and recombinant antigen for calibration, and is now available commercially as the Spec cPL (canine pancreas-specific lipase) assay. The Spec cPL assay has a sensitivity of 63.6% in necropsied dogs with macroscopic evidence of pancreatitis; however, the specificity was not reported in that study.[11] To the authors’ knowledge, there is only 1 recently published study reporting the specificity of the Spec cPL assay for diagnosing pancreatitis in 40 dogs without clinical or histopathologic evidence of the disease.[12] The reported specificity of the Spec cPL in that study was 97.5% (95% confidence interval [CI], 86.8–99.9%).[12]

The study objectives were to determine the sensitivity and specificity of several serum markers including Spec cPL, lipase, amylase, and cTLI for the diagnosis of pancreatitis in dogs using pancreatic histopathology on freshly procured pancreata from necropsied dogs as the “gold standard” of diagnosis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals

This study evaluated 70 client-owned dogs that were euthanized for a variety of reasons at the University of California, Davis, Veterinary Medical Teaching Hospital (UCD VMTH) between June 2007 and January 2009. Inclusion criteria for the study included the owners’ willingness to participate, the need to collect a serum sample from the dog within 24 hours of euthanasia, and the removal of the entire pancreas from each dog within 4 hours of euthanasia. Sixty-nine of 70 dogs had a full necropsy performed following removal of the pancreas and one dog had a pancreatectomy only following euthanasia. For all dogs in the study, a serum sample was obtained within 24 hours before euthanasia for serum biochemistry analysis, Spec cPL, cTLI, lipase, and amylase determination. All of the dogs’ owners signed an informed consent form, and the study was approved by the Animal Care and Use Committee at the UCD VMTH.

Histopathologic Evaluation

The entire pancreas and adjacent omental tissue was atraumatically removed from each dog within 4 hours of euthanasia. Sections of stomach, liver, and small intestine were routinely collected and evaluated in 44 (63%), 62 (89%), and 51 dogs (73%) respectively, and processed according to the UCD VMTH necropsy protocol. These tissues were collected either on the same day as the pancreas or within 24 hours of the pancreatectomy procedure. The right (duodenal) limb of the pancreas was tagged with suture material and abnormal gross abdominal lesions were recorded. The entire pancreas was placed in a large plastic container on a paper towel and immersed in 10% neutral-buffered formalin at a formalin to tissue ratio > 10 : 1. The pancreas was transversely sectioned into the left (gastric) limb, right limb, and body after at least 48 hours of fixation. For each limb of the pancreas, serial transverse sections were made every 2 cm and placed into several embedding cassettes. The tissues were paraffin embedded, routinely processed, and 5-μm sections were cut and stained with hematoxylin and eosin (HE) according to standard protocols. All the histopathology specimens were reviewed and scored in a blinded fashion by 1 board-certified veterinary pathologist (BGM).

A semiquantitative histopathologic grading scheme based on a published scoring system[13] was established that reflected the severity of acute and chronic pancreatic lesions. A histopathologic diagnosis of pancreatitis was defined as the presence of one or more histopathologic changes in one or more sections: suppurative inflammation, acinar cell necrosis, peripancreatic fat necrosis, mononuclear inflammation, or fibrosis. These changes may or may not have included hemorrhage, interstitial edema, atrophy, or cellular degeneration. Each transverse section was individually scored for all 9 parameters on a scale from 0 to 3. A score of 0 indicated that none of the sectional surface area was affected by that particular histopathologic change. Scores of 1, 2, and 3 had < 10%, 10–40%, and > 40% of the examined surface area affected by the specific lesion parameter, respectively. Additional histopathologic changes not captured by this scoring system were also recorded for each pancreas (eg, nodular hyperplasia and neoplasia).

Quantitative Analysis

For each lesion parameter, the mean cumulative score incorporated the sum of the scores for each parameter for all the pancreatic sections (left, body, and right limb), divided by the total number of sections. Gastric, hepatic, and intestinal biopsies were reviewed by the same veterinary pathologist (BGM) in a blinded fashion. Morphologic diagnoses were assigned for the alimentary and hepatic tissues based on standards set forth by the World Small Animal Veterinary Association (WSAVA) Gastrointestinal Standardization Group[14] and WSAVA Liver Standardization Group, respectively.[15]

Hematology, Biochemistry, and Serological Markers for Pancreatitis

Serum samples were obtained ≤ 24 hours before euthanasia and stored at −20°C for up to 6 weeks before being transported to IDEXX Reference Laboratories in West Sacramento, CA, for analysis. All serum biochemistry parameters including lipase1 and amylase2 were determined for all dogs on an automated chemistry analyzer.3 The Spec cPL assay4 is a quantitative microtiter plate ELISA and concentrations were measured on a microplate reader.5 Serum cTLI6 concentrations were determined on an automated immunoassay analyzer.7 Twenty-four of 70 dogs had a complete blood count (CBC) performed at the UCD VMTH hematology laboratory within 24 hours of euthanasia and 16 dogs had a CBC performed between 24 hours and 1 week before euthanasia. The remaining 30 dogs either did not have a CBC or had one completed ≥ 1-week before euthanasia.

Antemortem Abdominal Ultrasound

Digital ultrasound images performed within 24 hours of euthanasia were available in 8 of 70 dogs, and were reviewed in a blinded fashion by a board-certified veterinary radiologist (EGJ) at the conclusion of the study to determine whether ultrasonographic evidence of pancreatitis was present or not. All ultrasound studies were performed using a Phillips HDI 5000 ultrasound machine8 utilizing a blended 8–5 MHz ultrasound probe. Specific ultrasonographic features that were utilized to document evidence of pancreatitis included two or more of the following features: pancreas larger in axial height than the adjacent duodenal axial height; hyperechoic mesentery surrounding the pancreas; hypoechoic pancreas compared to the surrounding mesentery; and hyperechoic pancreas relative to normal liver.[16]

Statistical Analysis

Differences in each of the 9 pancreatic pathology parameters (as measured by mean and maximum score values) between the left, right, and main bodies of the pancreas were tested using a Friedman two-way analysis of variance. Pearson's correlation coefficient (r) was calculated for every diagnostic test and pathology metric score combination to assess the magnitude of its linear correlation. Positive correlation reflected a diagnostic test whose value increased with increasing evidence of pancreatic injury; negative correlation reflected an inverse relationship. Histopathology score was also correlated with diagnostic test values, and a sensitivity analysis that sequentially dropped individual metrics from the composite metrics to see if correlation substantially changed, was also performed.

Pancreatic tissue was subclassified by score as reflective of either group (1) no evidence of pancreatitis (score of 0), group (2) mild pancreatitis (score of 0.01–9.0), group (3) moderate pancreatitis (score of 9.01–18.0), and group (4) severe pancreatitis (score of 18.01–27). Because of the small number of dogs with severe pancreatitis, groups 3 and 4 were combined (moderate to severe pancreatitis) to increase the power of the study for this comparison. The ability of each diagnostic test to discriminate between dogs in these 3 subcategories was assessed by calculating the sensitivity and specificity (and the respective 95% CIs) of the test, utilizing conventional laboratory cutoff values to distinguish normal and abnormal values. Three sets of group comparisons were made for the diagnostic test analyses: Group 1 versus Group 2; Group 1 versus Group 3; and Group 1 versus Groups 3 and 4 combined. P-values less than .05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Dog breeds were diverse and included 10 mixed breed dogs, with no apparent breed predilections noted. Thirty-four of the dogs included in this study were male (3 intact, 31 neutered) and 36 were female (5 intact, 31 spayed). The dogs’ ages ranged from 9 months to 15 years (mean 8.5 years, median 9.2 years).

For analysis of pancreatitis, the best composite statistic used was the cumulative score (mean) and this metric was utilized for all of the statistical comparisons. The data set was analyzed in several other ways by calculating the average of the mean in which the mean score was calculated for the left limb, body, and right limb; the average of the maximum score in which the highest score identified for all transverse sections in each limb was calculated for the left limb, body, and right limb; and the maximum score for each parameter for the entire pancreas. Results of these composite scoring systems were similar to those derived from the cumulative score; hence the cumulative score was used for all comparisons.

Histopathologic evidence of mild pancreatitis was observed in 56 of 70 dogs, moderate pancreatitis in 6 of 70 dogs, and severe pancreatitis in 1 of 70 dogs. Seven of 70 dogs had no histopathologic evidence of pancreatitis. Virtually all the 63 dogs with pancreatitis had histopathologic features of “acute” and “chronic” pancreatitis concurrently, and only 5 dogs had histopathologic features of acute pancreatitis alone. For this reason, the statistician and coauthor on the article (PHK) concurred that the groups be combined to increase the power of our study. For the 7 dogs with no histopathologic evidence of pancreatitis, 6 had a Spec cPL < 200 μg/L and 1 had a Spec cPL between 200 and 400 μg/L. For the 56 dogs with histopathologic evidence of mild pancreatitis, 32 had a Spec cPL < 200 μg/L, 12 had a Spec cPL between 200 and 400 μg/L, and 12 had a Spec cPL > 400 μg/L. For the 7 dogs with histopathologic evidence of moderate to severe pancreatitis, 2 dogs had a Spec cPL < 200 μg/L, and 5 dogs had a Spec cPL > 400 μg/L.

Eleven of the 17 dogs (65%) with Spec cPL concentrations > 400 μg/L had clinical signs supportive of pancreatitis (vomiting, anorexia, weight loss), and 6 of the 17 dogs (35%) had physical examination findings of abdominal pain. No clinical signs or physical examination findings were reported in the medical records for 2 dogs. Six of these 17 dogs had macroscopic evidence of pancreatitis, with 4 of 6 having histopathologic evidence of moderate to severe pancreatitis. Additional pathological findings for 16 of 17 dogs included disseminated neoplastic disease (9), hepatic cirrhosis (2), immune mediated hemolytic anemia (1), glomerulonephritis (1), disseminated cryptococcosis (1), pneumonia (1), and myelodysplasia (1).

Serum amylase was the least sensitive serological assay for the diagnosis of pancreatitis with a sensitivity of 7% (95% CI = 2–17%) for mild pancreatitis, and 14% (95% CI = 0.4–58%) for moderate to severe pancreatitis, whereas serum lipase was the least specific serological assay (42.9% [95% CI = 10–82%]) for dogs with mild and severe pancreatitis (Table 1). The Spec cPL and serum lipase assays were the most sensitive assays for the diagnosis of moderate to severe pancreatitis (71% [95% CI = 29–96%]); however, the Spec cPL was more specific than all the other assays (100% [95% CI = 59–100%] and 86% [95% CI = 42–100%]) using a cutoff of 400 and 200 μg/L, respectively.

Table 1. Performance characteristics for the Spec cPL, cTLI, lipase and amylase in 70 dogs with and without histopathologic evidence of pancreatitis.
TestOutcomeSensitivity (%) (95% CI)Specificity (%) (95% CI)
  1. CI, confidence interval; cTLI, canine trypsin-like immunoreactivity.

Spec cPL
(Cutoff >400 μg/L)Mild pancreatitis21 (12–34)100 (59–100)
Moderate to severe pancreatitis71 (29–96)100 (59–100)
(Cutoff >200 μg/L)Mild pancreatitis43 (30–57)86 (42–100)
Moderate to severe pancreatitis71 (29–96)86 (42–100)
cTLI (>35 μg/L)Mild pancreatitis30 (19–44)100 (59–100)
Moderate to severe pancreatitis29 (4–71)100 (59–100)
Amylase (>1240 U/L)Mild pancreatitis7 (2–17)100 (59–100)
Moderate to severe pancreatitis14 (0.4–58)100 (59–100)
Lipase (>750 U/L)Mild pancreatitis54 (40–67)43 (10–82)
Moderate to severe pancreatitis71 (29–96)43 (10–82)

The correlation coefficients for the Spec cPL assay was highest for suppurative inflammation (0.45), peripancreatic fat necrosis (0.43), acinar cell necrosis (0.42), and edema (0.35); histopathologic features most consistent with acute pancreatitis (Table 2). Histopathologic features consistent with chronic pancreatitis such as fibrosis (0.34), atrophy (0.24), and lymphocytic inflammation (0.16) had a poorer correlation with the Spec cPL. Despite these differences, the correlation coefficients for each of the 9 histopathologic features were highest for the Spec cPL assay compared to cTLI, serum amylase, and lipase (Table 2).

Table 2. Correlation coefficients for diagnostic tests and pancreatic histopathology using cumulative score in 70 dogs with and without histopathologic evidence of pancreatitis.
 Suppurative InflammationAcinar cell NecrosisHemorrhageInterstitial EdemaPeripancreatic Fat NecrosisLymphocytic InflammationFibrosisAtrophyCellular DegenerationPancreatitis
  1. cTLI, canine trypsin-like immunoreactivity; cPL, canine pancreatic lipase.

  2. a

    One of the 63 dogs with histopathologic evidence of pancreatitis had a cTLI concentration of 1.3 μg/L, diagnostic for exocrine pancreatic insufficiency. The same dog had a Spec cPL of 232 μg/L. Seven other dogs with pancreatitis had cTLI concentrations between 2.5 and 5.0 μg/L.

Spec cPL0.450.420.210.350.430.160.340.240.210.45
cTLIa0.080.060.090.060.05−0.27−0.15−0.26−0.12−0.10
Amylase0.300.330.070.090.250.010.210.03−0.090.19
Lipase0.060.02−0.020.010.00−0.010.100.05−0.060.03

Twelve of 70 dogs had macroscopic evidence of pancreatitis at necropsy, including hemorrhage (8), edema (5), pancreatic nodules/plaques (6), a firm pancreas (4), hyperemia (2), pancreatic necrosis (1), and peripancreatic fat necrosis (3). The 12 dogs with macroscopic evidence of pancreatitis had significantly higher histopathologic scores for pancreatitis (P < .0001) compared to the 58 dogs without macroscopic evidence of pancreatitis. In addition, the Spec cPL was significantly higher in these 12 dogs compared to the other 58 dogs without macroscopic evidence of pancreatitis (P = .014). Five of 12 dogs with macroscopic evidence of pancreatitis had a histopathologic diagnosis of moderate to severe pancreatitis, whereas 7 of the 12 dogs had a histopathologic diagnosis of mild pancreatitis. Spec cPL was < 200 μg/L in 4 of 12 dogs, between 200 and 400 μg/L in 2 of 12 dogs, and > 400 μg/L in 6 of 12 dogs. Serum lipase activity was also significantly higher in the 12 dogs with macroscopic evidence of pancreatitis (P = .0005), although only 4 of these 12 dogs had abnormally increased serum lipase activity. In contrast, serum amylase activity and cTLI were not significantly different between the 2 groups of dogs (P = .17 and P = .12, respectively), and 4 of 12 and 5 of 12 dogs had abnormally increased serum amylase activity and cTLI concentrations, respectively.

Digital ultrasound images of the pancreas were available for interpretation in 8 of 28 dogs, and all of these dogs had ultrasonographic evidence of pancreatitis. In addition, all 8 dogs had histopathologic evidence of pancreatitis (3 dogs with mild pancreatitis and 5 dogs with moderate to severe pancreatitis). The Spec cPL was < 200 μg/L in 2 of these dogs, between 200 and 400 μg/L in 2 of these dogs and > 400 μg/L in 4 of these dogs. One dog with a histopathologic diagnosis of moderate pancreatitis had a Spec cPL of 76 μg/L, and 1 dog with a histopathologic diagnosis of mild pancreatitis had a Spec cPL of 77 μg/L.

Liver biopsies were examined in 12 dogs with Spec cPLs > 400 μg/L, and with histopathologic evidence of mild pancreatitis. Hepatic lesions in these dogs included: vacuolar hepatopathy (n = 9), lymphoma (n = 3), extramedullary hematopoiesis (n = 2), hepatocellular necrosis (n = 2), nodular hyperplasia (n = 2), hepatic congestion (n = 2), multifocal fibrosing portal lymphocytic hepatitis (n = 1), histiocytic sarcoma (n = 1), hepatocholangiocarcinoma (n = 1), metastatic islet cell carcinoma (n = 1), and hemorrhage (n = 1). Seven dogs had multiple lesions. The dog with metastatic islet cell carcinoma had pancreatic islet cell carcinoma and was found to have mild pancreatitis histopathologically. Intestinal biopsies were evaluated in 8 of these 12 dogs. Three of these dogs had abnormal intestinal pathology characterized by mural lymphoma of the duodenum (n = 1), mild segmental, plasmacytic enteritis (n = 1), severe necrosuppurative serositis (n = 1), mild focal acute hemorrhage of the ileum (n = 1), and crypt ectasia (n = 1). Two dogs had multiple lesions. Gastric biopsies were evaluated in seven of these 12 dogs, and abnormal findings were only evident in 1 dog that had evidence of severe submucosal edema of the stomach. Neoplasia in the pancreas was identified in 9 dogs: islet cell carcinoma (3), lymphoma (2), osteosarcoma (1), adenocarcinoma (1), mammary carcinoma (1), and transitional cell carcinoma (1).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The Spec cPL was the most sensitive and specific test compared to all other serological markers assessed, with a sensitivity ranging between 43 and 71% for dogs with mild to severe pancreatitis, using a cutoff value of 200 μg/L. The sensitivity decreased for dogs with mild pancreatitis (21%) when the cutoff was increased to 400 μg/L; however, the specificity increased from 86 to 100% using a cutoff of 400 μg/L. The stringent histopathological definition of “normal pancreas” (ie, no histopathological changes in any examined section) resulted in the classification of relatively few dogs as being normal (7 of 70). The high specificity might be partially attributable to this relatively small population of “normal animals”. The sensitivity findings in dogs with moderate to severe pancreatitis are similar to results reported in a recent study evaluating the sensitivity of the cPLI in 22 dogs with macroscopic and histopathologic evidence of pancreatitis following necropsy[11]; In that study, the cPLI had the highest sensitivity for dogs with macroscopic evidence of pancreatitis (64%) compared to other serological markers.[11] The specificity of the Spec cPL has been published in 1 study to date, and the test was found to be highly specific (98%) in 40 dogs with no histopathologic evidence of pancreatitis.[12] Our findings parallel those of Neilson-Carley et al, and the specificity of 100% (CI = 59–100%) using a cutoff of 400 μg/L suggests that false-positive test results are uncommon with the test.

The lower sensitivity for the Spec cPl in dogs with mild pancreatitis is likely a reflection of the stringent semiquantitative histopathologic scoring system that defined mild pancreatitis based on the presence of one or more lesion parameters affecting <10% of the surface area of the sections examined. It is plausible that the histopathologic definition of mild pancreatitis, as used in this study, is a broad one that includes many clinically normal animals. A second likely reason for the lower sensitivity of the Spec cPL observed in our study was the inclusion of pancreatic histopathologic features that are most consistent with chronic pancreatitis (lymphocytic inflammation, fibrosis, and atrophy) in our scoring system. A recent study of 14 cases of histopathologically confirmed chronic pancreatitis in dogs supports this hypothesis, as the sensitivity for the cPLI in that study ranged from 58% (95% CI 34–79%) to 26% (95% CI 10–51%) depending on the cutoff used.[17] Our semiquantitative scoring system was selected to avoid biasing our results toward dogs with only acute pancreatitis (suppurative inflammation, pancreatic necrosis, peripancreatic fat necrosis, and edema). Our study did confirm, however, that the correlation coefficients for lymphocytic inflammation, fibrosis, atrophy, and hemorrhage were lower than that for suppurative inflammation and pancreatic/peripancreatic necrosis. Pancreatic leakage of lipase would be less likely to be associated with pancreatic fibrosis and atrophy;[12] however, these correlations should be interpreted cautiously given that most of the variation in histopathologic scores cannot be explained by the serological tests. A correlation of 0.45 (suppurative inflammation) indicates that approximately 20% of the variation in histopathologic score can be explained by Spec cPL.

In a previous study of 22 dogs with macroscopic evidence of pancreatitis, a common macroscopic manifestation of pancreatitis was peripancreatic fat necrosis revealed by the presence of saponified fat.[11] The most common histopathologic findings included neutrophilic pancreatic infiltration (19 of 22 dogs), pancreatic necrosis (16 of 22), and peripancreatic fat necrosis (18 of 22).[11] That study also revealed the Spec cPL to have a higher correlation with the histopathologic pancreatitis activity index, a scoring system constructed for correlation with acute pancreatitis, compared to a histopathologic pancreatitis chronicity index.[11] These combined findings suggest that the Spec cPL may be more useful for identifying dogs with acute pancreatitis compared to chronic pancreatitis.

All 12 dogs that had a Spec cPL > 400 μg/L and histopathologic evidence of mild pancreatitis had histopathologic evidence of a hepatopathy, and only one of these dogs had macroscopic evidence of pancreatitis. Likewise, 3 of 12 dogs that had a Spec cPL > 400 μg/L and histopathologic evidence of mild pancreatitis had histopathologic evidence of intestinal lesions (mild to moderate plasmacytic enteritis, B-cell lymphoma, or necrosuppurative serositis with crypt ectasia). Further studies are warranted to assess Spec cPL concentrations in dogs with extrapancreatic disease, in particular dogs with liver disease, given the findings of increased serum lipase activity in dogs with liver disease.[5] Ultrasound evaluations were only performed in 8 of 70 dogs within 24 hours of euthanasia, and caution should be heeded in overinterpreting the findings given the limitations of a retrospective examination of still ultrasound images and the small number of dogs evaluated via ultrasound. Nevertheless, ultrasound evaluations did correctly diagnose pancreatitis that was ultimately confirmed via histopathology in all 8 dogs, and the Spec cPL was > 400 μg/L in 4 of these dogs and between 200 and 400 μg/L in 2 dogs. Two dogs with ultrasonographic evidence of pancreatitis and histopathologic evidence of mild and moderate pancreatitis had Spec cPLs of 77 and 76 μg/L, respectively.

The cTLI assay has been described as a highly sensitive and specific test for the assessment of exocrine pancreatic insufficiency,[18] however, the performance characteristics of the assay are suboptimal for the diagnosis of pancreatitis,[7] and the low sensitivity of this assay was confirmed in this study. Measurement of serum amylase activity was the least sensitive test for the diagnosis of mild or moderate to severe pancreatitis; however, the high specificity for this test and the cTLI can be explained by the relatively low numbers of dogs lacking histopathologic evidence of pancreatitis, and the fact that these tests were within the reference interval for all these dogs. Measurement of serum lipase activity was the second most sensitive serological marker for the diagnosis of pancreatitis, and the higher sensitivity ranging from 54 to 71% should be interpreted cautiously, given the nature of the study and the finding of a relatively large number of dogs with extrapancreatic disease.

A limitation of this study was that these dogs were necropsied for a variety of reasons, and that clinical inferences (weight loss, inappetance, vomiting, etc) that often are associated with pancreatitis could have been because of concurrent disease. The sensitivity data must be interpreted cautiously because of the relatively small number of dogs with histopathologic evidence of moderate to severe pancreatitis, and because of the broad inclusion of histopathologic features that are associated with both acute and chronic pancreatitis. Likewise, the relevance of mild pancreatic inflammation observed histopathologically may not reflect clinical disease, and the histopathologic findings of pancreatic suppurative inflammation, pancreatic necrosis, and peripancreatic fat necrosis may be more optimal histopathologic markers to assess pancreatitis.[12]

In summary, the Spec cPL demonstrated the best overall performance characteristics (sensitivity and specificity) relative to the other serological tests for the diagnosis of pancreatitis in dogs, with pancreatitis being defined histopathologically as opposed to clinically. Caution should be heeded in interpreting the specificity results, because these were based on 7 normal pancreata only. The Spec cPL appears more suitable for the diagnosis of acute pancreatitis in dogs, although the test showed higher correlations with all of the pancreatic histopathologic features compared to the other serological tests. The sensitivity of the cTLI was inferior to that of serum lipase, underscoring the limitations of the cTLI for the diagnosis of canine pancreatitis. Further prospective studies are warranted to evaluate the diagnostic yield of combining abdominal ultrasound with determination of Spec cPL in dogs with suspected pancreatitis, in an effort to improve the combined sensitivities when both tests are performed in parallel.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This research was supported in part by an ACORN grant from the American Kennel Club Canine Health Foundation. The authors acknowledge the technical support of IDEXX Laboratories, West Sacramento, CA, and the invaluable input provided by Dr Jane Robertson, IDEXX Laboratories, West Sacramento, CA.

Conflict of Interest Disclosures: None of the authors declares a potential conflict of interest in this manuscript.

Notes
  1. 1

    Lipase method: 1,2-diglyceride, Genzyme Diagnostics, Cambridge, MA

  2. 2

    Amylase method: 2-chloro-4-nitrophenol, Genzyme Diagnostics, Framingham, MA

  3. 3

    Olympus AU5400 Chemistry Analyzer, Olympus American Inc, Center Valley, PA

  4. 4

    Spec cPL Test, IDEXX Laboratories Inc, Westbrook, ME

  5. 5

    Max Endpoint ELISA Microplate Reader, Molecular Devices Inc, Sunnyvale, CA

  6. 6

    Canine TLI, Siemens Healthcare Diagnostics Products Limited, Llanberis, UK.

  7. 7

    Immulite 2000 Automated Immunoassay Analyzer, DPC Cirrus Inc, Flanders, NJ

  8. 8

    Philips Healthcare, 3000 Minuteman Road, Andover, MA

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • 1
    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). J Am Vet Med Assoc 1998; 213:665670.
  • 2
    Cook AK, Breitschwerdt EB, Levine JF, et al. Risk factors associated with acute pancreatitis in dogs: 101 cases (1985–1990). J Am Vet Med Assoc 1993; 203:673679.
  • 3
    Simpson KW, Simpson JW, Lake S, et al. Effect of pancreatectomy on plasma activities of amylase, isoamylase, lipase and trypsin-like immunoreactivity in dogs. Res Vet Sci 1991; 51:7882.
  • 4
    Polzin DJ, Osborne CA, Stevens JB, et al. Serum amylase and lipase activities in dogs with chronic primary renal failure. Am J Vet Res 1983; 44:404410.
  • 5
    Quigley KA, Jackson ML, Haines DM. Hyperlipasemia in 6 dogs with pancreatic or hepatic neoplasia: Evidence for tumor lipase production. Vet Clin Pathol 2001; 30:114120.
  • 6
    Mansfield CS, Jones BR, Spillman T. Assessing the severity of canine pancreatitis. Res Vet Sci 2003; 74:137144.
  • 7
    Archer FJ, Kerr ME, Houston DM. Evaluation of three pancreas specific protein assays, TLI (trypsin-like immunoreactivity), PASP (pancreas specific protein) and CA 19-9 (glycoprotein) for use in the diagnosis of canine pancreatitis. Zentralbl Veterinarmed A 1997; 44:109113.
  • 8
    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; 64:12371241.
  • 9
    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; 67:175182.
  • 10
    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; 63:722727.
  • 11
    Steiner JM, Newman S, Xenoulis P, et al. Sensitivity of serum markers for pancreatitis in dogs with macroscopic evidence of pancreatitis. Vet Ther 2008; 9:263273.
  • 12
    Neilson-Carley SC, Robertson JE, Newman SJ, et al. Specificity of a canine pancreas-specific lipase assay for diagnosing pancreatitis in dogs without clinical or histologic evidence of the disease. Am J Vet Res 2011; 72:302307.
  • 13
    Newman SJ, Steiner JM, Woosley K, et al. Histologic assessment and grading of the exocrine pancreas in the dog. J Vet Diagn Invest 2006; 18:115118.
  • 14
    Day MJ, Bilzer T, Mansell J, et al. Histopathological standards for the diagnosis of gastrointestinal inflammation in endoscopic biopsy samples from the dog and cat: A report from the World Small Animal Veterinary Association Gastrointestinal Standardization Group. J Comp Pathol 2008; 138(Suppl 1):S1S43.
  • 15
    WSAVA Liver Standardization Group. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. 1st ed. Philadelphia, PA: Saunders Elsevier; 2006, p. 41124.
  • 16
    Hecht S, Henry G. Sonographic evaluation of the normal and abnormal pancreas. Clin Tech Small Anim Pract 2007; 22:115121.
  • 17
    Watson PJ, Archer J, Roulois AJ, et al. Observational study of 14 cases of chronic pancreatitis in dogs. Vet Rec 2010; 167:968976.
  • 18
    Williams DA, Batt RM. Sensitivity and specificity of radioimmunoassay of serum trypsin-like immunoreactivity for the diagnosis of canine exocrine pancreatic insufficiency. J Am Vet Med Assoc 1988; 192:195201.