Validation of a point-of-care assay for simultaneous quantification of equine adrenocorticotropic hormone and corticotropin-like intermediate peptide.

Overview

• This study evaluated a new point-of-care immunoassay designed to simultaneously measure two hormones, ACTH and CLIP, in horses to diagnose pituitary pars intermedia dysfunction (PPID), comparing its accuracy and precision to the standard laboratory test.

Background

• Equine pituitary pars intermedia dysfunction (PPID) is a common endocrine disorder in horses, diagnosed primarily through measuring plasma levels of adrenocorticotropic hormone (ACTH).
• The standard chemiluminescent immunoassay used for ACTH measurement can cross-react with corticotropin-like intermediate peptide (CLIP), another hormone elevated in PPID, potentially affecting accuracy.
• A novel point-of-care (POC) assay was developed to simultaneously quantify both ACTH and CLIP in plasma samples, potentially improving diagnostic precision and convenience.

Study Objective

• To validate and compare the performance of the new POC-ACTH/CLIP immunoassay against the established chemiluminescent immunoassay for ACTH (iACTH) in horses.
• To assess the accuracy, precision, bias, and linearity of the POC assay under various conditions including seasons and after stimulation testing.

Methods

• Study Design: Prospective observational study involving 28 university-owned adult horses; 13 diagnosed with PPID and 15 without PPID.
• Sample Collection: Resting plasma hormone levels were measured multiple times across all four seasons over one year, and once after thyrotropin releasing hormone (TRH) stimulation to provoke ACTH secretion.
• Testing: Each sample was analyzed by both the POC-ACTH/CLIP assay and the reference chemiluminescent iACTH assay.
• Statistical Analysis:
  • Used Spearman correlation to evaluate the strength of association between the two assays.
  • Bland-Altman plots assessed agreement and bias (difference in hormone values between tests).
  • Intra-assay and inter-assay coefficients of variation (CV) were calculated to evaluate repeatability and reproducibility of the POC assay.
  • Linearity (ability to produce accurate results across a range of concentrations) was evaluated by regression analysis.

Results

• Correlation: There was a strong positive correlation between POC-ACTH/CLIP and the reference iACTH assay overall (correlation coefficient r ≥ 0.94; P < .001), and specifically in PPID horses (r=0.93; P < .001).
• Bias:
  • The POC assay tended to show a greater bias (difference from reference values) during autumn, measuring on average 33.6-35.6 pg/mL higher with wide variability (±89.5-96 pg/mL), compared to smaller biases in winter and spring (1.6-11.1 pg/mL ± 8.4-29.6 pg/mL).
  • At higher hormone concentrations (≥ 90 pg/mL), the bias increased substantially (mean difference 87.8 ± 116.4 pg/mL), indicating less precise agreement at elevated levels.
• Precision:
  • Intra-assay CV (repeatability within the same run) was 8.47%, indicating acceptable repeatability.
  • Inter-assay CV (reproducibility across separate runs) was 14.1%, considered acceptable for clinical assays.
• Linearity: The assay demonstrated excellent linearity (R² = 0.991), suggesting accurate measurement across a broad range of hormone concentrations.

Conclusions

• The novel POC-ACTH/CLIP immunoassay performs well in a field setting and correlates closely with the established chemiluminescent assay for ACTH measurement.
• It offers the advantage of simultaneously measuring both ACTH and CLIP, which may improve diagnostic insight in PPID since both hormones are elevated.
• Despite overall good agreement, clinicians should be cautious interpreting results during autumn and at higher hormone levels due to increased bias and variability.
• The assay’s acceptable precision and strong linearity support its use as a convenient and reliable diagnostic tool for equine PPID monitoring.