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Powerful Picture

     Colour Graphics Analyser

Analysing ToolAnalysing Tool FREEWARE


  Directions for use  
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Directions for use

Defining measurement profile (etalon), exemplified for nuclear staining of estrogen receptor with DAB.
Load image. Button "Select and move targets" is active. The is seen.
The image size can be modified using to fit the screen.
Define targets using right mouse click. After defining a few, their size and line colorcan be in place already. The target moved using right mouse click (drag and drop) or cleared (see ).
To establish the first profile try to define as much targets as possible in different colors, covering entirely the staining results from negativity to positivity.

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.

Defining measurement profile (etalon), exemplified for nuclear staining of Ki67 with DAB.

Load image and define targets Select sort method Red The targets are sorted and you can identify the treshold value using tool

After that the colour group borders are defined and the settings are verified using "Calculate". In the "Calculate" window please define Type 2 as positive (double click at - sign). The etalon can be saved (button "Save setup all readers in Sort method window). For repeated measurments the etalon can be corected as

A simple morphometry (relative area estimated by point counting) using Random targets. The microvascular density measurment according to the Chalkley.

This method (Chalkley count) is estimates the relative area of microvessels (immunostained with anti CD34 antibody). At least three "hot spots" within the section are captured at 200x magnification. After image is loaded, the are placed (use "Modify number of random targets" before - the appropriate number is 100-203, depends from image size and shape). The target size should be smaller (5-8), please modify in Use sort method Red and 2 categories (positive-negative) etalon e.g. Ki67 estimate before. The percentage of brown area appears in T2 category. Repeat for three images an average the percentage. This approach can be applied also for more complicated tasks