Open tools for color analysis.  Panel  "Sort methods" serves to select and switch the algorithms of sorting. Panel "Target colours" shows the average colors within targets (currently unsorted).

Select sort method. There are essentially two kinds of sort methods available: simple and two step. The simple methods e.g. (red, Green, Blue) sort targets according to the amount of the key colour component. In two steps sorts first a treshold discriminating between positive and negative staining (Hue, Brown, BN, GB) is to be estimated. In the second step Lightness or Saturation are applied to ensure fine sorting for semiquantitative assessment. For this purpose serves the another tool: Html view. In following example we will use sort method H+L.
First the sort with default value H=0 is performed

The targets are sorted but brown color does not appear at one side. Using Html view we will be able to set the proper value for H. Balance 0 is not OK as can be seen here:

If balance is set to 2,0 the discrimination is much better:

The target colors sorted this way show the gradient from dark brown to dark blue:

Now the adequacy of sorting values can be checked using the tool enabling to identify the sorted targets within original image.

If satisfactory result is obtained, the next step is to define the categories of staining in the window by clicking appropriate squares in sorted Target colours. Up to six categories can be defined. If the category border is not identical with the treshold value, such a category is automatically divided. See an example with four categories of positivity and one negative. This is used for histoscore counting.

The numbers in lower row indicate the numbers of squares. The white label in Targets colour window indicatees the H treshold value. Now the targets can be calculated.

A formula for histoscore counting can be defined using "Score". A category is first defined as positive or negative (double click) and the the index is defined. In this case the final score is 0-400. The formula is 4*percentage of strongest positivity + 3*percentage of strong positivity + 2*percentage of weak positivity + 1*percentage of borderline positivity  (cit.)

The remarks can be added and the complete result can be saved including them.

The defined categories and calculation rules represent a "Profile" or "etalon". These are specific for different sort methods, can be saved under different names from window " " for different purposes. Furthermore, they can be modified, but still are backward compatible as will be shown here:

Let us open another image.

Opening the tools:

Now we open and load the saved profile (etalon) . The targets are automatically sorted and after Calculate also labeled. The white labels mean the target colour does not fit within a defined range.

This is to be corrected as seen below. Note the numbers of targets appearing now in lower row of color group labels. After another calculate all targets are labeled properly.

An updated profile is to be saved. Usually after 10 to 20 measurments a definite profile is set up.