IMAGE-ANALYSIS QUANTIFICATION OF THE MILES ASSAY

The Miles assay for vascular permeability has high intra- and interass ay coefficients of variation (CVs). Quantification, usually by dye ext raction and spectrophotometry, is time consuming. In this study, quant ification by this means was compared with image analysis using the Oly mpus CUE-2 Image Analyzer (version 4). The test substance was recombin ant human vascular permeability factor (rhVPF). The quantification pro cess took approximately 10 min with image analysis. Formamide extracti on and spectrophotometry required 1 hr of preparation, 4-6 days of inc ubation, and 1 hr for filtration and spectrophotometry. Between assay CVs ranged from 0 to 30% for spectrophotometry, but were all <10% for image analysis. The sensitivity (2SD above the negative control mean) of the image analysis approach was 64 +/- 25 ng/mL, whereas for spectr ophotometry it was 65 +/- 29 ng/mL. Interanimal CVs for rhVPF at 200 a nd 1000 ng/mL were 15% and 26% when assessed by spectrophotometry and 7% and 22%, respectively, by image analysis. The R(2) value for the co rrelation of image analysis with spectrophotometry was 81.4%. Test sub stances injected close to the spine evoked a greater permeability resp onse than those injected laterally: at 200 ng/mL p = 0.005, at 1000 ng /mL p = 0.1 (unpaired t tests).