Development of a Method to Evaluate Cat Dander Levels by Safranin-O Staining

Abstract

Fel d 1 is primarily responsible for allergic symptoms. However in cat exposure rooms replicating home exposure, Fel d 1 levels can vary greatly over time making it difficult to predict patient exposure. ELISA assays delay the ability to regulate allergen levels in a timely fashion. The purpose of this study is to investigate dander counting by staining, light microscopy, and image processing and correlate counts to Fel d 1 levels. This could allow prompt measurements and tighter allergen level control. Dander samples were obtained after shaking cat bedding for 1 minute every 15 minutes over 1 hour. Dander was collected by gravity for 15 minutes on isopore and glass fiber filters at 30, 45 and 60 minutes. Fel d 1 collected on glass fiber filters was quantified using ELISA (Indoor Biotechnologies). Isopore filters were stained with Safranin-O (0.01%) for a minimum of 60 minutes and visualized by light microscopy. Ten images were captured on each quadrant of the filter. Particle count, area and perimeter of detected particles were obtained by computer analysis using ImageJ Software. Particle size distribution, the area ratio of detected particles, and particles/mm2 were then calculated. The area ratio of particles detected as well as the particle count per mm2 increased from 15 minutes to 1 hour. A linear correlation was found between Fel d 1 levels and area ratio (R2=0.810). The results of this study could be useful for monitoring allergen concentrations in cat challenge chambers.