The purpose of this program is to quantity the representative surface of the analyzed objects of an image and measure the intensity of their staining. It means the percentage of positive objects of the predetermined region of analysis can be expressed. This analysis algorithm is used to quantify IHC nuclear or cytoplasmic staining presenting segmentation difficulties. The surface approach is an approximation of an object-based approach.
The protocol :
The different stages of image analysis using the Immuno Surface algorithm are as follows :
- Identification of the color plane
The algorithm carries out automatic color deconvolution (DAB/H or AEC/H). This deconvolution can subsequently be adjusted by the user.
- Image segmentation
Image segmentation enables the identification of the objects that are to be analyzed without seeking to individualize them. This process is carried out automatically and can subsequently be refined using a cursor.
- Definition of staining positivity thresholds
Morphomathematical tools enable the segmentation to be refined. The pixels of the segmented surface are then placed into four categories for which positive thresholds must be determined. This particular approach does not involve individualizing the analyzed objects.
The results are surface-expressed. This surface is distributed according to the staining intensity of objects that represent it, as per the four predetermined categories, from negative to intensity levels 1+, 2+ and 3+. The results also express the surface area represented by each of the four categories of intensity. As the analyzed objects are not individualized, an overall result is obtained.
The proportion of the positive surface, which is the ratio of the stained surface against the total surface, then determines the staining index.