ImmunoRatio: an ImageJ plugin for analyzing
ER, PR, and Ki-67 immunohistochemistry

About   |   Screenshots   |   Download   |   How to use   |   Algorithm description   |   Authors

ImmunoRatio is an open source plugin for the ImageJ image analysis software. The plugin: 1) automatically segments diaminobenzidine-stained (DAB) and hematoxylin-stained nuclei regions from any number of source images, 2) for each image, calculates the percentage of DAB-stained nuclear area over total nuclear area (=labeling index), and 3) averages the labeling indexes and generates a pseudo-colored result montage showing the area segmentation (see example screenshots).

Please note: ImmunoRatio is also available as a free, online web application at http://jvsmicroscope.uta.fi/immunoratio/, which requires no software installation. In addition, the application is described in a journal article published in the Breast Cancer Research.

Screenshots

Example result #1  Example result #2  Example result #3

Download

ImmunoRatio plugin (version 1.0c, 14.2.2011)

You will also have to download and install a Java Runtime Environment (JRE 1.6 or newer), ImageJ (version 1.42m or newer), and the LOCI Bio-Formats plugin package (loci_tools.jar). Place the downloaded JAR files into your ImageJ plugin directory and restart the program. The source code for ImmunoRatio is included within the JAR file, which can be extracted using a conventional ZIP archiver.

License: ImmunoRatio is licensed under the GNU General Public License. See the license.txt for more details.

Disclaimer: ImmunoRatio is a diagnostic aid for personnel trained to score immunostained samples. Analysis results should always be interpreted together with the pseudo-colored images and the original slides. The developers of ImmunoRatio expressly disclaim any and all liability for any loss or damage that may arise directly or indirectly by using the software or content available on this web site. By downloading the software, you expressly agree and consent to the foregoing terms and conditions.

How to use

Details on how to: prepare samples, define optimal image acquisition settings, calibrate the analysis to match your laboratory-specific assessments, use the blankfield image, use the result correction equation, and so on, please see the Help section of ImmunoRatio web application.

Interactive usage with ImageJ

  1. Important: prior to first analysis, measure the scale (in pixels / micron) of your microscope setup. This can be done, for example, by using a measuring glass, or, by using the ImmunoRatio web application's Scale Finder. If the scale is not known, the analysis will most likely fail.
  2. Open any number of microscope images within ImageJ. For example, an image triplet from the same sample:

    Image triple
  3. Start the ImmunoRatio plugin. The following user interface is opened:

    ImmunoRatio GUI

    Blankfield correction image: if empty, artificial background correction is applied (not recommended)
    Log file: stores the averaged results into a user-specified text file
    Result image directory: stores the result montages into a user-specified directory
    Source image copy directory: stores copies of the original source images into a user-specified directory
    Brown threshold adjustment: fine-tunes the DAB component thresholding
    Blue threshold adjustment: fine-tunes the hematoxylin component thresholding
    Source image scale: the image scale (in pixels / micron)
    Result correction equation: a 3rd degree polynomial that is applied to the result labeling index (has no effect if a=0, b=0, c=1, and d=0); the default polynomial is precalibrated to match visual cell counting (see ImmunoRatio web application for details)
    Ask sample identifier: rather than using the image title/filename as the sample ID, asks for a string
  4. When you are ready to start the analysis, click the OK button.
  5. Once the analysis is finished, a montage image consisting of every analyzed image and their result average will appear:

    Result montage

Macro usage (batch mode)

ImmunoRatio can also be used in batch mode via the ImageJ macro language. The calling syntax is the following (using the default result correction):

    run(
        "ImmunoRatio ",
        "source_images=C:\\sample1.jpg,C:\\sample2.jpg,C:\\sample3.jpg " +
        "blankfield_image=C:\\blankfield.tif " +
        "log_file=C:\\log.txt " +
        "result_image_directory=C:\\results\\ " +
        "source_image_copy_directory= " +
        "brown_threshold_adjustment=0 " +
        "blue_threshold_adjustment=0 " +
        "scale_pixels_per_um=4.60 " +
        "result_correction_coefficient_a=0.00006442 " +
        "result_correction_coefficient_b=-0.0019840 " +
        "result_correction_coefficient_c=0.61100000 " +
        "result_correction_coefficient_d=0.43210000 " +
        "sample_identifier=example_identifier "
    );

Algorithm description

ImmunoRatio algorithm

Authors

Vilppu Tuominen, MSc
Jorma Isola, prof., MD, PhD

Cancer Biology research group
Institute of Biosciences and Medical Technology (BioMediTech)
University of Tampere, Finland

Email: firstname.lastname@uta.fi

© Cancer Biology research group, Institute of Biosciences and Medical Technology (BioMediTech), University of Tampere BioMediTech UTA