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Partial Analytical Validation of microRNAs-148a and 375 for Detecting Pancreatic Injury in Healthy Dogs and Dogs With
Caylen Erger1, Kyan Thelan Strong1, Harry Cridge1
1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA.
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 = 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.
Results:
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).
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.
Insights
microRNA (miRNA) assays for canine pancreatitis are analytically valid, but hemolysis can interfere. miRNA-148a levels were lower in dogs with pancreatitis, suggesting potential as a biomarker.
Area of Science:
- Veterinary diagnostics
- Biomarker discovery
- Molecular biology
Background:
- MicroRNAs (miRNAs) show promise as canine pancreatitis biomarkers due to their correlation with acinar cell injury.
- Analytical validation and interference effects of potential miRNA biomarkers in dogs remain largely uncharacterized.
Purpose of the Study:
- To develop and analytically validate assays for serum miRNA-148a and miRNA-375 in dogs.
- To compare serum miRNA-148a and miRNA-375 levels between healthy dogs and those with pancreatitis.
Main Methods:
- Quantified circulating miRNAs (miRNA-148a and miRNA-375) from serum of 40 healthy and 40 pancreatitis-affected dogs.
- Assessed analytical validity, including linearity (dilutional parallelism) and precision (intra- and inter-assay coefficients of variation).
- Investigated the interference effects of Intralipid, bilirubin, and hemoglobin on the miRNA assays.
Main Results:
- Both miRNA assays demonstrated analytical validity with high linearity (O/E ratios ~100%) and low coefficients of variation (<3.2%).
- Hemolysis significantly impacted both assays, causing false increases (p < 0.003).
- miRNA-148a was significantly reduced in dogs with pancreatitis (mean 192 copies) compared to healthy controls (mean 2760 copies; p < 0.0001), while miRNA-375 showed no significant difference (p = 0.94).
Conclusions:
- The developed miRNA assays are analytically valid for canine serum.
- Hemolysis, a common issue in pancreatitis cases, can interfere with results, necessitating careful sample handling.
- Suppressed levels of miRNA-148a in pancreatitis-affected dogs warrant further investigation into its role as a potential diagnostic or prognostic biomarker.
