Materials and Methods

Animals—Dogs that were euthanatized or that died because of various medical conditions unrelated to this study were enrolled. The first set of dogs was from Bay Area Veterinary Specialists, a large referral hospital in Northern California. For client-owned animals, informed consent for entry into the study was obtained. Some of the dogs were surrendered by the owners prior to euthanasia or death, and when the attending clinician carefully determined that each dog had a poor prognosis or poor quality of life, it was euthanatized. The remaining dogs in this study were from an animal shelter and were scheduled to be euthanatized after being deemed unadoptable by the shelter staff and following permission from the director of the shelter. Humane euthanasia protocols were followed for each dog that was euthanatized at the hospital, and the standard humane operating protocols were used at the shelter. At both institutions, euthanasia was performed via IV lethal injection of pentobarbital. The study protocol was approved by a client-owned-animal investigations review committee at Bay Area Veterinary Specialists.

Twenty dogs from a previous study¹⁰ of serum markers of pancreatitis were also included. These dogs were selected from a group of 208 dogs submitted 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 during necropsy. To qualify for inclusion in the present study, the dogs were also required to have at least 1 clinical sign consistent with pancreatitis, including vomiting, diarrhea, anorexia, or abdominal pain. The dogs that were included had clinical signs that ranged from 1 to all of the aforementioned clinical signs. However, the severity of these clinical signs could not be ascertained from the data available to the present investigators.

Evaluation and sample collection—Dogs from the referral hospital received a physical examination, and some dogs underwent additional diagnostic testing, including thoracic and abdominal radiography, abdominal ultrasonography, and diagnostic laboratory analyses. Results of diagnostic tests were used to assess the overall health of the dogs and to help determine a definitive diagnosis for dogs that were not healthy. Dogs from the shelter were briefly examined prior to euthanasia. Each was sedated with acetylpromazine, ketamine, and xylazine (only some dogs) administered IM prior to evaluation as part of shelter standard operating procedure because many were aggressive and had severe signs of anxiety. Doses of the aforementioned drugs were as follows: for dogs weighing < 11.4 kg, 0.5 mg of acetylpromazine, 45 mg of ketamine, and 10 mg of xylazine; for dogs weighing 11.4 to 34.1 kg, 10 mg of acetylpromazine and 100 mg of ketamine; and for dogs weighing > 34.1 kg, 15 mg of acetylpromazine and 150 mg of ketamine.

For all dogs from the hospital as well as from the shelter, blood samples for a CBC, serum biochemical analysis including amylase and lipase activity, and cPSL concentration assay^a were obtained prior to euthanasia (but after sedation, in the shelter dogs). The entire pancreas was removed from all dogs after euthanasia or death.

Histologic evaluation—The pancreas was harvested from each dog (from the referral hospital and shelter and in the previous study) within 6 hours after death and placed in 10% formalin for at least 48 hours prior to processing. The pancreases of dogs in the present study were sectioned in 1-cm-thick slices, and those of dogs from the previous study were sectioned in 2-cm-thick slices. Sections were routinely processed, stained with H&E, and evaluated and scored by 1 board-certified pathologist (SJN), who used a published grading protocol.¹¹ Briefly, scores were assigned as follows: 0, lesion absent; 1, < 10% of the section affected; 2, 10% to 40% of the section affected; and 3, > 40% of the section affected. Mean cumulative scores for each category were calculated by dividing the sum of the scores from each section by the number of sections evaluated. The number of sections evaluated for each pancreas ranged from 13 to 32, depending on the length of the respective pancreas. Each section of each pancreas was scored for the presence of neutrophilic infiltration, lymphocytic infiltration, pancreatic necrosis, peripancreatic fat necrosis, edema, fibrosis, atrophy, and hyperplastic nodules.¹¹

Statistical analysis—Dogs were divided into 2 groups for statistical comparison. One group consisted of dogs with a clinical diagnosis or macroscopic diagnosis of pancreatitis. The second group consisted of dogs with no clinical evidence of pancreatitis but that had conditions such as trauma, dystocia, anemia, neoplasia, severe osteoarthritis, high liver enzyme activities, leukocytosis, and hemoconcentration.

Nonparametric statistics were used to compare median MCSs for each histologic characteristic to determine whether a difference in scores existed between dogs with and without pancreatitis. A Mann-Whitney U test was performed when mathematically appropriate. When the median MCS for a group of dogs was 0, the Wilcoxon signed rank test was used instead. Significance was set at a value of P ≤ 0.05 for all analyses, which were performed by use of a statistical software package.^b

Once it was determined which histologic characteristics significantly differed between dog groups, an MCS of 0.0 was chosen for those characteristics associated with pancreatitis in which enzyme leakage would be expected. The specificity of the cPSL test was then calculated from the data for dogs with no clinical or histologic evidence of pancreatitis. A cPSL assay value > 400 μg/L is reportedly consistent with a diagnosis of pancreatitis.¹² Therefore, a cPSL assay value greater than this cutoff in a dog with no clinical or histologic evidence of pancreatitis was considered a false-positive result.

Results

Animals—Pancreata were obtained from 17 dogs (10 males and 7 females) from the referral hospital. Of these dogs, 16 were humanely euthanatized and 1 died of respiratory arrest after being hit by a car. The reasons for euthanasia of the other dogs included the following: hit by car (n = 3), dystocia (2), miscellaneous trauma (2), maxillary osteosarcoma (1), severe osteoarthritis (1), diabetic ketoacidosis (1), adrenal tumor (1), severe anemia (1), metastatic sarcoma involving the liver and spleen (1), pancreatic adenocarcinoma (1), and pancreatitis (2). Both dogs with pancreatitis had clinical signs that included vomiting, diarrhea, anorexia, and lethargy. Both were quiet during physical examination, and 1 dog had noticeable signs of discomfort when its abdomen was palpated. One of the dogs with pancreatitis had high serum alanine transferase and alkaline phosphatase activities. The clinical diagnosis of pancreatitis for both was based on clinical findings and abdominal ultrasonographic changes consistent with pancreatitis, including a hypoechoic pancreas and hyperechoic surrounding mesentery. The known ages of dogs from the referral hospital ranged from 3 months to 16 years (median, 8 years). Breeds included mixed breed (n = 9) and 1 each of Toy Poodle, Chow Chow, Cocker Spaniel, Shih Tzu, Chihuahua, Collie, Scottish Terrier, and Weimaraner.

Pancreata were obtained from 27 dogs (15 males and 12 females) from the animal shelter. One dog was noticed to be emaciated during physical examination and, upon review of laboratory results, was found to have diabetes mellitus. Seven shelter dogs appeared healthy upon physical examination but had minor abnormalities in their laboratory results, including mild nonregenerative anemia (n = 3), mild leukocytosis with mature neutrophilia (2), mild increase in liver enzyme activities with mild nonregenerative anemia (1), and hemoconcentration (1). The remaining 19 dogs appeared healthy upon physical examination and had unremarkable results of hematologic and serum biochemical analyses. Reproductive status was not reported as it was unknown for many females. Definitive ages were also not known for most dogs; thus, the age for many of these dogs had to be estimated by the shelter staff on the basis of dental characteristics and overall appearance. Ages ranged from approximately 3 months to > 5 years. Breeds included pit bull-type dog (n = 17), mixed breed (4), Rottweiler (2), German Shepherd cross (2), German Shepherd (1), and Plotthound (1).

Clinical signs compatible with pancreatitis were present in 20 of the 22 dogs with macroscopic evidence of pancreatitis from the previous study. This included vomiting in 18 of 22 (82%) dogs, abdominal pain in 10 (45%), anorexia in 13 (59%), and diarrhea in 8 (36%). Nine of the 22 (41%) dogs underwent ultrasonography, and evidence of pancreatitis was detected in 6.¹⁰

MCS comparisons—Results were summarized for statistical comparisons of MCSs between dogs with a clinical diagnosis or macroscopic diagnosis of pancreatitis and those with no clinical evidence of pancreatitis (Figures 1–8). Differences between the 2 groups in median MCS were significant for each of the following histologic parameters between dogs with and without pancreatitis: neutrophilic infiltration (P < 0.001), pancreatic necrosis (P < 0.001), peripancreatic fat necrosis (P < 0.001), pancreatic edema (P = 0.016), fibrosis (P < 0.001), atrophy (P < 0.001), and nodules (P < 0.001). They were not significant (P = 0.128) for lymphocytic infiltration.

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Mean cumulative histologic scores for degree of neutrophilic inflammation in pancreatic tissues (range, 13 to 32 sections/pancreas) from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS differs significantly (P < 0.001) between groups. Scores were assigned as follows: 0, lesion absent; 1, < 10% of the section affected; 2, 10% to 40% of the section affected; and 3, > 40% of the section affected. Horizontal line indicates median score.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Mean cumulative histologic scores for lymphocytic inflammation in pancreatic tissues from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS does not differ significantly (P = 0.128) between groups. See Figure 1 for key.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Mean cumulative histologic scores in pancreatic tissues from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS differs significantly (P < 0.001) between groups. See Figure 1 for key.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Mean cumulative histologic scores for peripancreatic necrosis in pancreatic tissues from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS differs significantly (P < 0.001) between groups. See Figure 1 for key.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Mean cumulative histologic scores for pancreatic edema in pancreatic tissues from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS differs significantly (P = 0.016) between groups. See Figure 1 for key.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Mean cumulative histologic scores for pancreatic fibrosis in pancreatic tissues from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS differs significantly (P < 0.001) between groups. See Figure 1 for key.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Mean cumulative histologic scores for pancreatic atrophy in pancreatic tissues from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS differs significantly (P = 0.004) between groups. See Figure 1 for key.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Mean cumulative histologic scores for pancreatic hyperplastic nodules in pancreatic tissues from dogs with pancreatitis (n = 22) and dogs with other diseases (42). The MCS differs significantly (P < 0.001) between groups. See Figure 1 for key.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.302

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Specificity of the cPSL assay—Because leakage of pancreatic enzymes would not be expected with pancreatic fibrosis, atrophy, or the presence of nodules and because there was no significant difference in median MCS for lymphocytic infiltration between dogs with and without pancreatitis, these histologic characteristics were not considered when selecting the dogs for determination of the specificity of the cPSL assay. Therefore, a pancreas was classified as having no histologic evidence of pancreatitis when the MCSs for neutrophilic infiltration, pancreatic necrosis, peripancreatic fat necrosis, and edema were 0.0.

Forty of 42 (95%) dogs with no clinical evidence of pancreatitis had an MCS of 0.0 for neutrophilic infiltration, pancreatic necrosis, peripancreatic fat necrosis, and edema. These dogs were used to calculate the specificity of the cPSL assay. Thirty-eight of 40 (95%) dogs with no evidence of pancreatitis had a serum cPSL concentration within the reference interval (< 200 μg/L), resulting in a specificity of 95.0% (95% confidence interval, 83.1 to 99.4%) at this concentration. Thirty-nine of 40 (98%) dogs had a cPSL value < 400 μg/L, and thus the specificity associated with the diagnostic cutoff for pancreatitis was 97.5% (95% confidence interval, 86.8% to 99.9%).

Discussion

Although histologic assessment is often referred to as the gold standard for the diagnosis of pancreatitis, there are no established criteria by which to categorize a pancreas as having histologic evidence of pancreatitis. By comparing the degree of histologic changes in pancreata, through use of an established grading system, between dogs with clinical or macroscopic evidence of pancreatitis and dogs that were healthy or had a disease other than pancreatitis, we determined which characteristics differed between the 2 groups.

Not surprisingly, there was a significant difference in the amount of neutrophilic infiltration, pancreatic and peripancreatic fat necrosis, and edema between the 2 groups of dogs. However, it was interesting that there were also differences in the amount of atrophy, fibrosis, and hyperplastic nodules. This would suggest that some of the dogs with pancreatitis likely had previous episodes of pancreatitis or ongoing chronic pancreatitis resulting in more chronic and reparative lesions in their pancreata. However, one would not expect pancreatic leakage of lipase to be associated with pancreatic fibrosis, atrophy, or hyperplastic nodules; accordingly, no histologic cutoffs were established to exclude dogs with only these types of pancreatic lesions. In another study,¹³ hyperplastic nodules were detected with increased frequency in older versus younger dogs and their presence did not correlate with the presence of other lesions, suggesting that such hyperplastic nodules are associated with age and do not indicate previous or current pancreatic inflammation. Two dogs in the group without pancreatitis had diabetes mellitus: 1 had a large amount of fibrosis and atrophy in its pancreas, and the other had fibrosis and hyperplastic nodules. Both dogs had serum cPSL concentrations within the reference interval.

The group of dogs used to calculate the specificity of the cPSL assay included dogs with various diseases, none of which had clinical signs or histologic evidence of pancreatitis. In a clinical setting, dogs for which the cPSL assay would be used would most likely have clinical signs of pancreatitis, including vomiting, diarrhea, hematochezia, inappetence, and abdominal pain. Therefore, pancreatitis would not likely be a major differential diagnosis for the dogs that were enrolled the present study. It is important to consider that the specificity of 97.5% was calculated for dogs that would not likely undergo testing for pancreatitis in a clinical setting. On the basis of our study findings, the cPSL assay appeared to be specific in dogs with a lack of histologic lesions consistent with pancreatitis and for which pancreatitis was not considered a major differential diagnosis. This limitation of the study could not be overcome because removal of the entire pancreas in such clinical patients is only possible for dogs that die because of their disease process, which would introduce yet another variable.

The severity of the clinical signs in the 20 dogs with pancreatitis was unknown. Unfortunately, these data were not available to the authors. All dogs in the pancreatitis group that were used for the study had at least 1 clinical sign of pancreatitis. Therefore, these dogs would likely warrant cPSL testing in a clinical setting.

An unexpected finding was that there was no significant difference in the median MCS for lymphocytic infiltration between dogs with and without pancreatitis. There were 11 dogs in each group with no lymphocytic inflammation in any of the tissue sections evaluated. In the group of dogs without pancreatitis, the highest MCS for lymphocytic inflammation was 0.9, which was assigned in a dog with pancreatic adenocarcinoma. The only other lesions in that dog's pancreas were fibrosis and hyperplastic nodules, and the dog had a serum cPSL concentration within the reference interval. The highest MCS from a dog with macroscopic pancreatitis and a clinical history of anorexia was 1.3. Similarly, that dog had no other lesions in its pancreas with the exception of fibrosis and hyperplastic nodules; however, its cPSL concentration was high at 812 μg/L. Lymphocytic infiltration would be expected to be present in dogs with chronic pancreatitis. Future studies are needed to determine the degree of lymphocytic infiltration that results in clinically important chronic pancreatitis and the sensitivity of the cPSL assay in dogs with chronic pancreatitis. However, findings of the present study suggested that mild lymphocytic infiltration did not result in an increase in cPSL concentration in dogs with no clinical signs of pancreatitis.

For a dog to be categorized as having no histologic evidence of pancreatitis, there had to be no neutrophilic infiltration, pancreatic necrosis, peripancreatic fat necrosis, or edema present in any of the pancreatic sections evaluated. Only 2 dogs without clinical evidence of pancreatitis were excluded on the basis of these criteria. One was a 4-month-old female mixed-breed dog that had been hit by a car; its MCS for peripancreatic fat necrosis was 0.1, and its serum cPSL concentration was 30 μg/L. The second was an 8-year-old male mixed-breed dog that had a hepatic and splenic sarcoma, and it had an MCS of 0.2 for both neutrophilic inflammation and peripancreatic fat necrosis and a cPSL concentration of 171 μg/L. Including these dogs would have actually increased the specificity of the cPSL assay slightly. However, the aforementioned histologic changes were considered important on the basis of previously defined criteria. Consequently, these dogs were excluded from the group categorized as having no histologic evidence of pancreatitis.

The reference interval for serum cPSL concentration is ≤ 200 μg/L, and the cutoff value for diagnosing pancreatitis is 400 μg/L.¹² In the present study, there was 1 dog with a serum cPSL concentration higher than the cutoff value of 400 μg/L that had an MCS of 0.0 for neutrophilic infiltration, pancreatic necrosis, peripancreatic fat necrosis, and edema. The only laboratory abnormality in that dog was hemoconcentration. The dog was a 3-year-old male German Shepherd cross from the animal shelter and had a serum cPSL concentration of 513 μg/L. The only histologic abnormalities in its pancreas were hyperplastic nodules, with an MCS for this category of 0.2. The reason for this discrepancy is unknown. It is possible, although unlikely, that a laboratory error occurred for the sample from this dog. No serum was available to repeat the cPSL analysis. Results of a previous study¹⁴ suggest that microscopic lesions are highly localized in the exocrine pancreas in pancreatitis. Therefore, an alternative possibility is that the dog in question had localized pancreatic lesions that were missed during sectioning of the pancreas for histologic analysis. There was an additional dog that fulfilled the study criteria of having no histologic evidence of pancreatitis and that had a cPSL concentration higher than the upper reference limit but lower than the cutoff for diagnosing pancreatitis. This additional dog had a serum cPSL concentration of 230 μg/L, the only laboratory abnormality was a leukocytosis, and the MCS for every histologic characteristic was 0.0.

The definition of specificity for any diagnostic test is the number of true-negative test results for the disease (ie, the number of dogs with a cPSL value less than the diagnostic cutoff value for pancreatitis of 400 μg/L that do not have the disease; n = 39) divided by the total number of dogs without the disease (40). Thus, the overall specificity of the cPSL assay in the present study for dogs with no clinical or histologic evidence of pancreatitis was 97.5%. Use of the reference interval for the specificity calculation instead would have resulted in a specificity of 95%.

The present study had some limitations. Part of the standard operating protocol for the shelter animals was to administer acetylpromazine and ketamine to all dogs for sedation along with xylazine for some, as many of these dogs were too aggressive to handle otherwise. This action did not appear to affect the results of the study, however. In addition, because a portion of the study involved dogs from a shelter, pit bull-type dogs were grossly overrepresented among shelter dogs; nevertheless, the same was not true of the dogs from the referral hospital.

Previous studies^10,c have shown that the serum cPLI and cPSL assays are sensitive for diagnosing pancreatitis. In the present study, only the specificity of the cPSL assay was determined in dogs without clinical or histologic evidence of pancreatitis. Additional studies are needed to determine the diagnostic specificity in dogs with clinical diseases for which pancreatitis is a differential diagnosis.