This repository offers an implementation of the WSICS algorithm, as described in "Stain Specific Standardization of Whole-Slide Histopathological Images" (10.1109/TMI.2015.2476509). WSICS can create templates from H&E stained images and use these to normalize other H&E stained images accordingly.

A precompiled binary is available for 64x Windows. Additionally a Docker Container can be build through the Dockerfile within the repository. These are both standalone, and don't require any additional work to function.

• Windows: https://github.com/computationalpathologygroup/WSICS/releases
• Docker: https://hub.docker.com/r/diagnijmegen/wsics

This project aims to provide source code that is compatible with both Windows and Linux. However, Linux systems may vary in terms of available packages, which is why we don't officially support or provide binaries for any specific distribution.

In order to compile the WSICS binary, the following prerequisites must be met:

• ASAP
• BOOST
• OpenCV

Due to the utilization of the ASAP image reading capabilities, all of its dependencies are required as well.

WSICS can be called through a CLI and accepts whole slide images in a tiled image format, or as a flat patch. The image reading is provided by the ASAP project, and thus provides any format that it does as well. WSICS attempts to locate tiles or static images that don't just contain background, if no tiles or static images are discovered that can be utilized for processing, adjusting the --background_threshold parameter can control the strictness of this process.

The normalization process requires that a template image is converted to a CSV file with relevant paramaters. Using another WSI directly isn't supported yet. Please see the --output_template parameter for the creation of a template file.

The containerized version of WSICS relies on volumes to access the required files and images. In order to access images and export results, a volume must be mounted through the -v option for docker. An example can be seen below:

docker run -v [local directory]:/data/ diagnijmegen/wsics --input /data/[image] --template_output /data/[template name]

It is possible for the algorithm to utilize a large amount of memory, which can result in a segfault error for Docker. To resolve this, the required training size can be diminished, or the pool of memory increased for Docker.

In order to execute the algorithm, the input parameter can be set with a file or directory path. If a directory path is offered, the algorithm will attempt to normalize all readable files within that directory. The output commands are interpreted based on the input, if a file is inserted, the output paths will be considered file paths as well, and vice versa for directories.

-i, --input [file or directory path]

If a directory is used as an input origin, the filenames themselves will be used to output the resulting normalized whole-slide images. To customize this output, a prefix or postfix can be set through the corresponding parameters.

--prefix [prefix string]
--postfix [postfix string]

To output normalized images, the image_output parameter must be set. This also outputs the LUT table as a tif file. If only the LUT table is required, the lut_output parameter can be set.

--image_output [file or directory path]
--lut_output [file or directory path]

A whole-slide image can be used as a reference for the normalization, which allows the algorithm to transform the source image to closer resemble the reference used. To do this, a set of template parameters are created and exported into a CSV file. This template can be generated by setting the template_output parameter, and used by setting the template_input parameter.

--template_input [file path to a template CSV file]
--template_output [file path to an output location]

If one or multiple whole-slide images contain ink or other heavily represented artifacts, then the ink or k parameter can be set. This will reduce the chance of a patch being selected to collect training pixels for the algorithm, and thus potentially insuring a better result.

-k, --ink

Several steps of the normalization process are based on randomized processes. In order to still offer a deterministic execution, the BOOST Mersenne Twister implementation has been utilized as the random generator. Its seed can be set through the seed variable.

-s, --seed [positive integer]

The creation of the Look Up Table utilizes a Naïve Bayes classifier to determine the probabilities of a pixel belonging to a certain class. In order to train this classifier, pixels corresponding to the background, Eosine and Hematoxyline colored tissue is selected and added to a training set. The max_training and min_training parameters define the total size of the training set created and the minimum amount of selected pixels required to continue an execution.

--max_training [size as integer]
--min_training [size as integer]

The training pixels are selected from tiles that contain little to no background, this is done by calculating the amount of pixels that are near white or black. If this is higher than the percentage indicated by the background_threshold parameter, then the tile isn’t utilized for the selection of training pixels.

--background_threshold [positive float]

Additionally, the amount of detected ellipses are also considered when selecting tiles to extract pixels from. Normally this is calculated based on the tile size. However, it can also be set through the min_ellipses

The selection of Hematoxylin colored pixels is done by detecting ellipses within the tissue and then calculating the mean red density value of the HSD color space. The hema_percentile parameter then defines which ellipse mean is selected to serve as threshold for the selection of Hematoxylin pixels.

--hema_percentile [float value between 0 and 1]

The Eosin threshold is defined by collecting all pixels that aren’t considered as background or Hematoxylin stained, and then selecting the one that is nearest within the list, based on the percentage defined by the eosin_percentile parameter. The pixels passing the threshold are then utilized for the creation and collection of Eosin stained pixels.

--eosin_percentile [float value between 0 and 1]

Normally, tiles that contain few detected ellipses are skipped and ignored for the creation of the training set. However, it's possible to set a static lower limit for the amount of detection, such as when there's little actual tissue on the image. This can be done with the min_ellipses parameter.

--emin_ellipses [positive integer]