Materials and Methods
Serum samples for pancreatic lipase (DGGR substrate) analysis were obtained prospectively by convenience sampling of horses and donkeys presenting to Bell Equine Veterinary Clinic for investigation of gastrointestinal disease, where surplus blood taken for routine clinical evaluation of the cases was available. For the purposes of this study, gastrointestinal disease was defined as equids presenting with clinical signs of acute colic and/or acute diarrhoea. Serum was harvested from clotted blood samples collected from eligible equids on admission to the hospital; in some cases, serum was also collected on consecutive days when blood was being drawn for ongoing clinical management. Consent was obtained from the owners for the use of left-over biological material for research purposes, both by the owners signing the hospital consent form and verbally. Serum was acquired by centrifugation of clotted blood samples and submitted to the laboratory within 24 hours of collection. Ethical approval was provided by the CVS Ethics Committee (CVS-2022-005).
Where available, results of peripheral haematological and plasma biochemical analyses (including white blood cell and neutrophil counts, packed cell volume, total protein and albumin concentrations, and lactate concentrations) and peritoneal total nucleated cell counts and lactate concentrations for samples obtained on admission to the hospital were recorded. The duration of illness prior to admission and severity of abdominal pain on admission were recorded, as were the heart rate, results of FLASH abdominal ultrasonography (including presence or absence of distended loops of small intestine, thickened intestinal walls, excessive peritoneal fluid, gastric distension, abdominal masses, intestinal intussusception, evidence of colonic displacement and hepatic abnormalities), and volume of any gastric reflux (recorded as spontaneous reflux or reflux obtained on passage of a nasogastric tube). The severity of colic pain was categorised as mild, moderate, or severe, based on a behaviour-based simple descriptive scale (Mair and Smith 2005). Any other significant clinical history or physical examination findings were also recorded. Cases were categorised as medical colic (i.e. colic that resolved with non-surgical treatment), surgical colic / euthanased without surgery (surgical cases euthanased without surgery for financial or welfare reasons), colitis, peritonitis and others. The diagnosis of colitis was based on the typical clinical, ultrasonographic and clinical pathological findings including diarrhoea, tachycardia, fever, leukopenia, hypoproteinemia, etc (Shaw and Stämpfli 2018). The diagnosis of peritonitis was based on the presence of an elevated total nucleated cell count and elevated total protein concentration in the peritoneal fluid, with cut-off values for nucleated cell count of > 10 × 109/L and total protein concentration > 25 g/L (Brownlow et al 1981). The final diagnosis (including non-specific colic) and outcomes (survived, euthanased or died) were recorded.
Serum pancreatic lipase concentrations were recorded and categorised as normal (0-20 U/L), mildly elevated (21–49 U/L), moderately elevated (50– 199 U/L) or markedly elevated (> 200 U/L), as previously described (Johnson et al 2019). In addition, pancreatic lipase concentrations that were less or more than twice the upper reference limit (< or >2 x URL) were also recorded (as described by Lanz et al 2022). Continuous data were assessed for normality graphically and using Shapiro-Wilk tests. Median, interquartile range (IQR) and range were reported for non-normally distributed data. Categorical data are presented showing the count and percentage. Non-parametric Wilcoxon rank-sum tests and Kruskal-Wallis tests were used for continuous variables. Post-hoc analysis following a statistically significant Kruskal-Wallis was undertaken using a Dunn’s test. Chi-squared tests were used for categorical variables. Correlations between two continuous variables were assessed using Spearman’s rank correlation. Statistical significance was set at p < 0.05. Data were analysed using Stata 17.0.