Validation of a Dry-slide Immunoassay for Progesterone Analysis in Canine Plasma in a Clinical Setting

Background: The identification of canine ovulation is critical for successful breeding. Progesterone measurements are useful for identifying ovulation. Progesterone assays are also quantitative and easily accessed, making them valuable in veterinary practice.

Objectives: We aimed to validate a dry-slide immunoassay (DSI) for use in dogs, including a method comparison with the chemiluminescence assay (CLIA) and mass spectrometry.

Methods: Twenty-nine bitches were prospectively recruited. Accuracy, precision, interference, and stability were evaluated. Method comparison between DSI and CLIA and mass spectrometry was conducted, and bias was calculated.

Results: Repeatability was 8.0%-10.8%, and within-laboratory imprecision was 8.8%-11.1% for four concentration levels. Recovery under dilution was 61%-100%, and the method was linear to a concentration of ~50 nmol/L. Recovery after the addition of a high progesterone sample was 76%-83%. Minor changes were seen in one hemolytic and two lipemic samples. Storage at room temperature for 12-24 hours resulted in concentrations that were 57%-96% of the initial concentrations. For samples frozen at -80°C, the concentrations were reduced 17%-27%. There was a significant difference between results from the DSI and CLIA, and a proportional bias was seen when DSI was compared with mass spectrometry, where CLIA correlated better than DSI.

Conclusions: Precision and accuracy were acceptable. A proportional bias was seen between DSI and CLIA. A small amount of interference was seen with hemolysis and lipemia. Progesterone concentrations were decreased in samples stored at room temperature and -80°C. The results support the use of the DSI for ovulation timing but not for artificial insemination with frozen semen since progesterone concentrations might exceed the assay's linearity and precision limits.