QUANTIFICATION OF AREA PERCENTAGE OF IMMUNOHISTOCHEMICAL STAINING BY TRUE COLOR IMAGE-ANALYSIS WITH APPLICATION OF FIXED THRESHOLDS

Most image analysis systems (IAS) use black-and-white cameras. However , true color IASs are considered to be useful for quantification of im munohistologically stained structures. Using a true color IAS, we eval uated two methods of segmentation for quantification of area percentag e of staining: one using fixed, preset thresholds and one using thresh olds interactively set per image. Furthermore, the effect of shading c orrection pens evaluated, and measurements in both color and black-and -white mode were compared. The results of segmentation with fixed thre sholds did not differ significantly from those of control percentages, established by interactive morphometry using a grid, which served as reference. Interactive segmentation was significantly different from t he reference (t test, P =.0002). The effect of shading correction teas negligible. Measurements with and without this procedure correlated h ighly (r =.99, P <.002). Comparison of the results obtained in color a nd black-and-white mode showed a significant difference in the latter from the reference (t test, P =.005). We conclude that it is possible to quantify, in a reliable way, area percentage of positive staining u sing a true color IAS with application of a segmentation method with f ixed thresholds.