Scatter plots comparing labeling indexes defined by visual cell counting, non-calibrated ImmunoRatio, and calibrated ImmunoRatio. (a) The calibration was made using a training set of 50 samples, of which 25 were stained for Ki-67, 13 for progesterone receptor (PR), and 12 for estrogen receptor (ER). To achieve linear relation (dotted line), a correction function was defined by fitting a third degree polynomial (solid black line) to the training set. (b) The calibration was validated by using a separate test set of 50 samples (25 stained for Ki-67, 13 for PR, and 12 for ER). The validation test set included two outliers (marked as brown).