Evaluation of a new immunoassay for measuring equine serum insulin and plasma adrenocorticotrophic hormone: A method comparison study.

Overview

• This study evaluated new immunoassays specifically designed for measuring equine serum insulin and plasma adrenocorticotrophic hormone (ACTH) by comparing them to established assay methods.
• The goal was to assess how well these new assays agree with currently used methods for diagnosing common equine endocrine disorders such as insulin dysregulation (ID) and pituitary pars intermedia dysfunction (PPID).

Background

• Insulin dysregulation (ID) and pituitary pars intermedia dysfunction (PPID) are common hormonal disorders in horses that require accurate measurement of circulating insulin and ACTH levels for diagnosis.
• Multiple immunoassays exist to measure these hormones, but different assay platforms can yield variable results, complicating comparisons and clinical decision-making.

Objective

• The study aimed to compare new equine-specific immunoassays—VIDAS® Equine Insulin and VIDAS® Equine ACTH, both enzyme-linked fluorescent assays (ELFA)—to other established assays:
• Chemiluminescence immunoassay (CLIA) using IMMULITE 2000XPi
• Chemiluminescence enzyme immunoassay (CLEIA) with TOSOH Automated Immunoassay Analyser-360
• Equine insulin enzyme-linked immunosorbent assay (ELISA; Mercodia)
• The goal was to assess agreement between these assays to evaluate the utility of the new ELFA methods in clinical settings.

Methods

• Samples:
• Residual serum samples (n=242) and plasma samples (n=337) originally submitted to a commercial lab were used for insulin and ACTH testing.
• Subsets of samples were also tested with CLEIA (126 insulin, 76 ACTH) and ELISA (76 insulin).
• An additional set of plasma samples from 139 healthy horses was used to establish reference ACTH ranges using the ELFA assay.
• Assays were run simultaneously for direct comparison between methods.
• Statistical analyses included:
• Median differences with confidence intervals to compare hormone concentrations across assays.
• Correlation coefficients (R) to assess strength of relationship between assay pairs.
• Bland-Altman analysis to quantify bias and limits of agreement between assays.
• Three-way classification based on adapted diagnostic cut-offs to determine agreement in diagnosing PPID and identifying laminitis risk.

Results

• Median differences between insulin assays varied from about 1.6 to 11.3 μIU/mL depending on the assay pairs compared, indicating that the absolute values are not identical but are fairly close.
• Median differences for ACTH assays ranged from approximately 10.7 to 23.7 pg/mL between methods.
• Correlation analyses showed:
• Strong to very strong correlations (R > 0.8 to > 0.9) between insulin assays.
• Strong correlation between ELFA and CLEIA ACTH assays.
• Moderate correlations (R > 0.6) for other ACTH assay pairs.
• Bland-Altman plots revealed moderate biases and wide limits of agreement, indicating some variability between assays despite overall correlation.
• When applying adapted diagnostic thresholds for insulin and ACTH, three-way classification showed substantial agreement, suggesting the assays would lead to similar clinical interpretations.
• A reference range for plasma ACTH in healthy horses was successfully determined using the ELFA assay.

Conclusions

• The new equine-specific ELFA assays measure insulin and ACTH at different absolute values compared to other assays but generally agree well enough for clinical use.
• Assays have good agreement in classifying horses for PPID diagnosis and laminitis risk, meaning the new ELFA tests are reliable alternatives.
• The study provides useful reference ranges for plasma ACTH in healthy horses using the ELFA method.
• Limitations noted include that samples were collected between July and November and did not include ponies, which may affect broader applicability.

Implications for Veterinary Practice

• Veterinarians can consider using these new ELFA assays knowing that while absolute values differ slightly, diagnostic decisions regarding equine endocrine disorders are likely to be consistent.
• Understanding the variation between assays underscores the importance of using assay-specific reference ranges and cut-offs when interpreting insulin and ACTH test results.
• Availability of an equine-specific assay may improve diagnostic accuracy and comparability across laboratories.