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 immunohistologically stained structures. Using a true color IAS, we evaluated two methods of segmentation for quantification of area percentage of staining: one using fixed, preset thresholds and one using thresholds interactively set per image. Furthermore, the effect of shading correction was evaluated, and measurements in both color and black-and-white mode were compared. The results of segmentation with fixed thresholds 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 the reference (t test, P = .0001). The effect of shading correction was negligible. Measurements with and without this procedure correlated highly (r = .99, P < .001). Comparison of the results obtained in color and 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 using a true color IAS with application of a segmentation method with fixed thresholds.