Serum concentrations of canine pancreatic lipase immunoreactivity and C‐reactive protein for monitoring disease progression in dogs with acute pancreatitis

Abstract Background Reliable biomarkers for monitoring disease progression and management in dogs with acute pancreatitis have not been described. Objective To determine if serum concentrations of canine pancreatic lipase immunoreactivity (cPLI) and C‐reactive protein (CRP) can be used as biomarkers for disease progression in hospitalized dogs with acute pancreatitis. Animals Thirteen hospitalized dogs with acute pancreatitis diagnosed based on clinical signs, serum cPLI concentrations, and imaging findings were enrolled. Methods Serum cPLI and CRP concentrations were determined before and then daily during hospital management and 1 week after hospital discharge. Modified canine activity index (MCAI) and canine acute pancreatitis clinical severity index (CAPCSI) scores were calculated daily for each patient while hospitalized. Results The MCAI scores (P = .03) but not CAPCSI scores (P = .31) were significantly different between dogs that survived to discharge (n = 11) and those that did not (n = 2). Serum cPLI concentration was positively correlated with MCAI (rho = 0.42; P = .01). Serum CRP concentration also was positively correlated with the MCAI (rho = 0.42, P = .01). Conclusions Serum cPLI and possibly CRP could be used as objective biomarkers for clinical changes in hospitalized dogs with acute pancreatitis. Additional studies involving larger numbers of dogs would be warranted to evaluate the broader impact of these findings.

Between 27% and 58% of dogs with acute pancreatitis die from the disease or from secondary complications. [22][23][24][25] Some affected dogs are slow to recover or appear stagnant in their clinical condition while hospitalized. This variability makes it difficult to determine prognosis and represents a financial burden on clients who can be unsure whether or not to continue management.
Previous studies have attempted to identify independent predictors of death in dogs with acute pancreatitis. One study described 2 scoring systems that were shown to predict short-term mortality (ie, death within 30 days) of dogs with acute pancreatitis: the canine acute pancreatitis severity score (CAPS) and the simplified version of CAPS (sCAPS). 25 The CAPS score incorporates the presence of systemic inflammatory response syndrome (SIRS), coagulation disorders, increased serum creatinine concentration, and ionized hypocalcemia. The CAPS score has a sensitivity of 89% and specificity of 90% for prediction of short-term (ie, <30 days) death in dogs with acute pancreatitis. The sCAPS includes respiratory rate instead of SIRS, 25 and has a sensitivity of 96% and a specificity of 77% for predicting short-term death in dogs with acute pancreatitis. The modified canine activity index (MCAI) is based on the canine inflammatory bowel disease activity index (CIBDAI), which is an objective measure for assessing the severity of inflammatory bowel disease (IBD) in dogs. The index since has been successfully used for a wide variety of studies of IBD in dogs. 12,[26][27][28] Because acute pancreatitis shares many clinical signs with IBD, the CIBDAI also was considered a potential study tool to assess dogs with acute pancreatitis and thus the CIBDAI has been modified for use in dogs with pancreatitis in some previous studies (Joerg M. Steiner, personal communication, 2021). In addition to serum concentrations of CRP and cPLI, we used both of these scoring systems to standardize the clinical evaluation of dogs with acute pancreatitis.
Our objective was to evaluate whether serum CRP and cPLI concentrations are useful biomarkers for assessing clinical changes in hospitalized dogs with acute pancreatitis. We hypothesized that serum CRP and cPLI concentrations would decrease during hospitalization in association with clinical improvement as dogs recovered from acute pancreatitis, as assessed by the MCAI and the canine acute pancreatitis clinical severity index (CAPCSI).

| Case enrollment
The study was approved by MedVet's research department and each client signed an informed consent document before patient enrollment. A prospective cohort study was performed for dogs that presented to the MedVet Emergency or Internal Medicine Service that were diagnosed with acute pancreatitis. Inclusion criteria included dogs that presented with ≥2 clinical signs associated with acute pancreatitis (eg, vomiting, diarrhea, hyporexia, anorexia, abdominal pain), had abdominal ultrasound examination findings consistent with pancreatitis (eg, hyperechoic mesentery with hypoechoic pancreas), and had serum cPLI concentration >400 μL/L at the time of presentation (day 0). Dogs were excluded if they had ≥2 previous episodes of acute pancreatitis (either suspected or confirmed), were suspected to have chronic pancreatitis, had serum cPLI concentration ≤ 400 μL/L, or were moderately or severely anemic or became moderately or severely anemic during the study (hematocrit <25%). Anemic dogs were excluded because of concerns regarding the necessity of repeated blood sample collection for the purpose of the study. Dogs also were excluded if serum samples were missing from any corresponding hospitalization or re-evaluation day, or if the blood sample was mishandled or thawed at any time.

| Clinical data collection
Modified canine activity index (Table 1) and CAPCSI (

| Biomarker evaluation
Serum samples for cPLI and CRP were processed at the GI Lab at Texas A&M University. Serum samples were stored at 80 C until processing.
Serum cPLI concentration was measured using a commercial ELISA (Spec cPL, Idexx Laboratories, Westbrook, Maine) and concentrations >400 μg/L were considered diagnostic for pancreatitis. Serum CRP concentrations were measured using an ELISA (Tri Delta Diagnostics, Boonton, New Jersey). Serum concentrations >10 mg/L were considered to be abnormal.

| Statistical analysis
The normality assumption was assessed by calculating descriptive statis- Linear mixed-effects models were used to estimate nonparametric correlations, while adjusting for individual dog effects. All data were ranktransformed before statistical analysis and models were fitted with clinical scores as the dependent variables and biomarkers as independent variables. The data set was restricted to observations from the day of  including the MCAI on re-evaluation 7-10 days after discharge) was 6 (4-13) and 3 (0-10) for died/euthanized and discharged dogs, respectively (P = .03). The median (range) CAPCSI during hospitalization was 2 (0-5) and 1 (0-3) for died/euthanized and discharged dogs, respectively (P = .31). The MCAI clinical scores decreased over time in enrolled patients (Figure 1), but a clear temporal pattern for CAPCSI scores was not apparent (Figure 2).  The majority of surviving dogs had serum albumin concentrations that increased from the time of the initial diagnosis to re-evaluation.
In contrast, both nonsurviving dogs had serum albumin concentrations that decreased before death. Nonsurvivors had quantitatively lower serum albumin concentrations than the majority of surviving dogs ( Figure 5).
In the majority of surviving dogs, serum cPLI concentrations increased with increasing MCAI clinical scores ( Figure 6) and these variables were significantly correlated after adjusting for the repeated measures sampling design in the survivor group, and when combining both the survivors and nonsurvivors into a single group (Table 3). Of the 2 nonsurvivors, the dog that was euthanized had an improved MCAI severity score as the serum cPLI concentration decreased.
However, the dog that died initially had improvement in the MCAI severity score and a lower serum cPLI concentration, but then the MCAI severity score and serum cPLI concentration plateaued the last few days before death.
The CRP concentrations also increased with MCAI severity score in both survivors and nonsurvivors (Figure 7), but the positive correlation was not significant after adjustment for the repeated sampling design when evaluating the survivor group individually (P = .1). However, when evaluating all dogs together, a significant correlation was observed between serum CRP concentrations and the MCAI severity score (P = .01).
No relationship was apparent between serum albumin concentration and the MCAI in survivors (data not presented) but a significant positive correlation was observed between these variables in nonsurvivors (P = .03). In the patient that was euthanized, the serum albumin concentration improved initially but then continued to decrease despite a decreasing MCAI. In the patient that died, the serum albumin concentration fluctuated similar to the MCAI, until the day it died when the serum albumin concentration decreased substantially with a mild decrease in the MCAI.
No significant relationship was found between serum cPLI concentrations and CAPCSI (Figure 8), nor between serum CRP concentrations and CAPCSI (Figure 9) in both survivor and nonsurvivor groups, or when all dogs were evaluated together. A significant correlation also was found between the MCAI and serum CRP concentration when both the survivor and nonsurvivor groups were combined. Although serum CRP concentration appears to be a useful biomarker to identify clinical changes in hospitalized dogs with acute pancreatitis, the decrease in serum cPLI was more consistent on a daily basis as compared to serum CRP concentration.

| DISCUSSION
Additionally, unlike serum cPLI, serum CRP concentration was not significantly correlated with MCAI when evaluating the survivor group alone. Serum CRP concentration is affected by a wide variety of conditions of different organs, [6][7][8][9][10][11][14][15][16][17]19,21 whereas serum cPLI is specific for acinar cell damage and pancreatitis. Many dogs in our study had concurrent diseases, so it is difficult to determine whether serum CRP concentration was affected by acute pancreatitis or the concurrent conditions. Serum CRP concentration might be more useful in dogs with pancreatitis but without concurrent conditions or complications. However, it is not uncommon for dogs to have at least 1 other concurrent condition or systemic complication in addition to moderate or severe pancreatitis, making it difficult to determine a correlation between CRP and improving or worsening pancreatitis. Another possible contributing factor as to why serum CRP concentration was not significantly correlated with the MCAI in surviving dogs is duration of hospitalization. A previous study found that serum CRP concentration was not significantly different between the surviving and nonsurviving groups in dogs with acute pancreatitis until the third and fourth day of hospitalization. 31  Our results provide additional information for the management of hospitalized dogs with acute pancreatitis and suggest that serum cPLI and CRP concentration might serve as objective markers of disease progression. Consistently increasing serum cPLI, CRP concentration, or both despite aggressive management may help ease owner concern about discontinuing treatment efforts. Additionally, it might prompt clinicians to adjust their current management.
In conclusion, the MCAI improved over time in most hospitalized dogs with acute pancreatitis that survived to discharge, whereas the