The purpose of this program is to individually quantity the objects in an image and to measure the intensity of their staining. The density or percentage or positive objects can be expressed for a predetermined region of analysis. This algorithm is used to quantify IHC nuclear staining, typically Ki67, RO, RP, P53, etc.
The protocol :
For an IHC slide with DAB or AEC with hematoxylin counterstaining, Immuno Object enables positive objects to be quantified in accordance with the following protocol:
- Identification of the color plane
The algorithm carries out automatic color deconvolution (DAB/H or AEC/H). This deconvolution can be adjusted by the user.
- Image segmentation
For each color plane, image segmentation processes enable identification of the objects that are to be analyzed. Object segmentation, which is done in accordance with their staining intensity, is automatic and can subsequently be refined using a cursor.
- Post-processing and definition of staining positivity thresholds
The segmentation of objects can be refined via morphomathematical processing and items are placed into four categories by adjusting positivity thresholds.
Results are expressed in the number and percentage of objects for each of the four categories. They are compared with the total number of objects present in the region of analysis. Analyzed objects can also be individualized and described on a one-by-one basis.
Objects are categorized according to the average intensity of their staining and sorted into 2 categories, Positive and Negative, or into 4 predetermined categories: 0, 1+, 2+ and 3+. The proportion of counted positive objects, which is the ratio of the number of stained objects against the total number of objects, then determines the staining index.