Validation of a stall-side progesterone assay for use in equine reproductive management

Abstract
Background: Successful reproductive management in mares depends on timely, accurate assessment of progesterone. A stall-side test that delivers rapid, accurate results has potential to transform equine reproductive care. Objectives: Validate the analytical and clinical performance of a point-of-care progesterone assay, “Sidekick” lateral-flow device and reader, and its agreement with established methods (RIA and CLIA). Study Design: Retrospective cohort Methods: Analytical performance assessed serially diluted mare plasma (0–8 ng/mL) to estimate precision and percent recovery. Clinical accuracy assessed by comparison with RIA and CLIA on paired samples (n=19). Agreement evaluated by regression analyses and ordinal concordance across clinically relevant progesterone bands. Cross-reactivity with the synthetic progestin altrenogest tested at pharmacological levels. Results: Sidekick showed excellent recoveries (90–102%) across 4–8 ng/mL, strong repeatability (CV 4–15%), and high reproducibility between laboratories (CV 9.9%). Ordinal agreement with reference standards was near-perfect in spiked samples (κ = 0.94) and substantial in clinical samples versus both RIA (κ = 0.77) and CLIA (κ = 0.79). No cross-reactivity with altrenogest detected. Stall-side Sidekick results were delivered in under 10 minutes. Main Limitations: Observed bias potentially due to matrix differing between methods (plasma and serum). Narrow validated range could cause high concentrations being underestimated. RIA single replicate design required assumption about its short-term variability. RIA and CLIA assays not designed for equine use, but are utilised in absence of equine specific assay. Conclusions: In stall-side use, the Sidekick assay provided rapid, accurate, and clinically meaningful progesterone results in mares across 4–8 ng/mL, enabling on-the-spot decisions and reducing delays from sample processing, transport and result reporting. Strong agreement with reference methods, combined with immediate availability and specificity for endogenous progesterone, make it a powerful tool for real-time reproductive management.