Neighborhood Count
The last feature in the Phenoplex guided workflow is the Neighborhood count feature used for neighborhood analysis.
The analysis of the neighborhood is a quantification of biomarker positive neighbor objects around a user defined target cell within a radius(µm) of a target object.
This enables users to specify Targets as the center points for the analysis as well as neighboring objects. Targets and neighbors are defined in the Threshold Step in the Phenoplex Guided Workflow. Multiple Thresholds can be combined to add up to a specific phenotype. Say for example; CD3 CD4 CD45 are defined as specific T helper cells in the images. Only objects that are positive for all three biomarkers CD3, CD4 and CD45 will be selected and counted for the neighborhood analysis.
Threshold results must have been saved the database in order to be able to perform a neighborhood analysis.
To begin neighborhood analysis:
First, select Neighbor Counts from the left vertical menu in the Phenoplex Guided Workflow. The options New Target and New Neighbor enable the addition of targets and neighbors, which can then be used to begin populating the neighborhood matrix table. Start by selecting New Target. Choose Characteristics of the Target by clicking on the drop-down menu underneath:
Then select the biomarkers for the Target:
Specify the ROI in which the target and their neighbor(s) should be quantified.
This feature functions only if an ROI has been previously defined in the image, either manually or through the use of an APP.
To add the target as a new Row entry to the Neighborhood Table click “Done”.
Additional Targets can be added, or the neighbor columns can be populated. To open the neighborhood definition menu, select the Add a New Neighbor button.
Define the characteristics of the Neighbor by selecting options from the drop-down menu below.
Then define the Radius around the Target(s) in which the Neighbor should be quantified. This is done by clicking in the Min. Distance and Max. Distance Field to update the radius band.
To add the target as a new column entry to the neighborhood table, click Done.
To add all required neighbors and targets for the analysis, repeat the whole procedure. After completing the analysis of all required neighbors and targets, select Calculate. The progress bar below the Calculate button indicates that the process is still running if not filled from left to right.
Switch to other image results using the Filter by menu.
When a different image is selected, the table updates to display results from the newly chosen image.
To change to different statistical values in the table, use the drop-down menu.
The Save button saves the selection of Targets and Neighbors including distance and ROI selection as a preset to the Database.
To highlight a specific cell and see all of its target objects and neighbors within the defined radius, perform a double click.
Clicking on the yellow and magenta color field opens a color picker to update colors as needed by the user:
The color is only updated in the selected row and/or column, while all others stick to the default yellow and magenta colors. Unless specified differently by the user.
Any generated data can be selected in the matrix and copied to Excel or any other program using the Ctrl+C and Ctrl+V keyboard shortcut.