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

## TL;DR

This study validates miRNA-148a and miRNA-375 as potential biomarkers for detecting pancreatic injury in dogs, finding that miRNA-148a is reduced in dogs with pancreatitis.

## Contribution

The study provides analytical validation of miRNA assays and identifies hemolysis as a potential interferant in testing.

## Key findings

- miRNA-148a was significantly reduced in dogs with pancreatitis compared to healthy dogs.
- Hemolysis caused false increases in miRNA-148a and miRNA-375 assay results.
- miRNA-375 levels were not different between healthy and pancreatitis-affected dogs.

## Abstract

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.

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.

Circulating miRNAs were quantified from serum of healthy (n = 40) and pancreatitis‐affected dogs (n = 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.

For miRNA‐148a, the RI was < 33 000 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 < 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 = 0.003 – < 0.0001). miRNA‐148a was reduced in dogs with pancreatitis (mean 192 gene copies) compared with healthy controls (mean 2760 gene copies; p < 0.0001), but miRNA‐375 was not different between groups (p = 0.94).

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.