Partial Analytical Validation of microRNAs-148a and 375 for Detecting Pancreatic Injury in Healthy Dogs and Dogs With Pancreatitis.

Abstract

BACKGROUND: microRNAs (miRNA) have been proposed as biomarkers for pancreatitis in dogs due to their high peak concentrations and experimental correlation with acinar cell injury. However, analytical validation and the effect of interfering substances are unknown. OBJECTIVE: The study aimed to (i) develop and analyze the analytical validity of an assay for miRNA-148a and miRNA-375 in serum and (ii) compare miRNA-148a and miRNA-375 between healthy and pancreatitis-affected dogs. METHODS: Circulating miRNAs were quantified from serum of healthy (n&#x2009;=&#x2009;40) and pancreatitis-affected dogs (n&#x2009;=&#x2009;40). Reference intervals were established, and serum miRNAs were compared between groups. Linearity was assessed via dilutional parallelism (observed/expected ratio, O/E). Precision was assessed via intra- and inter-assay coefficients of variation (% CV). Intralipid, bilirubin, and hemoglobin were tested as interferants. RESULTS: For miRNA-148a, the RI was <&#x2009;33&#x2009;000 gene copies and mean O/E was 100.4%. Mean inter-assay and intra-assay % CV were 1.5% and 1.3% respectively. For miRNA-375, the RI was <&#x2009;4 gene copies and mean O/E was 100.1%. Mean inter-assay and intra-assay % CV were 3.2% and 0.5% respectively. Hemolysis led to false increases in both assays (p&#x2009;=&#x2009;0.003 - <&#x2009;0.0001). miRNA-148a was reduced in dogs with pancreatitis (mean 192 gene copies) compared with healthy controls (mean 2760 gene copies; p&#x2009;<&#x2009;0.0001), but miRNA-375 was not different between groups (p&#x2009;=&#x2009;0.94). CONCLUSIONS: The miRNA assays were analytically valid; however, hemolysis, a common interferant in pancreatitis cases, may impact test results. miRNA-148a was suppressed in dogs with pancreatitis. It is unclear whether this represents a cause or a consequence of the disease.