Algorithm 1.

The Macenko stain normalization algorithm.

1: Convert RGB to optical density (OD)

2: Remove pixels with negligible optical density

3: Apply SVD on the OD tuples and use the largest 2 values to create the SVD plane

4: Project data onto the plane and normalize to unit length

5: Calculate the angle ϕ of each point with respect to the 1st (or 2nd) SVD direction

6: Find the robust extremes (αth and (100−α)th percentiles) of the angle ϕ

7: Find the projection of the extreme values back to OD space

8: Use this projection as optical density matrix (ODM)

9: Calculate the individual stain concentrations (Ch and Ce) using the inverse of ODM

10: Find the robust maximum ((100 − α)th percentiles) of the individual stain concentrations Ch, Ce

11: Normalize and transform concentrations to OD space and then back to RGB using an H&E template