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10092 - PD-L1, Cell Lines, Cancer

INTRODUCTION
The availability of genetically defined reference materials, offers an industry standard for development and quality control of IHC assays, directly, thereby improving the accuracy and reproducibility. Using engineered cell lines as quality control material to assess the performance of IHC assays eliminates the variability associated with patient-derived reference standards.

This APP can be used for quality control of PD-L1 strong positive, intermediate positive, weak positive and negative cell line material to ensure that each cell line block complies with the expected PD-L1 expression before the material is used as reference standard for IHC assays. The APP quantifies the PD-L1 expression in each cell line core and relates this expression to a known reference value for each cell line expression level.

KEYWORDS
PD-L1, cell line, quality control, reference standards, image analysis.

METHODS
The first image processing step involves a segmentation of all nuclei in the ROI [see EXAMPLES: figure 2 and 3]. This is done by using an HDAB-DAB color deconvolution band to detect positively stained nuclei and the Haematoxylin-HDAB color deconvolution band to detect negative nuclei. The color deconvolution bands are input to a K-means classifier.  A method for nuclei separation which is based on shape, size and nuclei probability is used, employing a fully automated watershed-based nuclei segmentation technique. Then the positive nuclei are subdivided into three categories based on staining intensities.  As a post-processing step, nuclei areas that are too small are removed. The image obtained after post-processing [see e.g. EXAMPLES: figure 3] is used for quantifying the PD-L1 expression in each cell line core based on the H-score. As a final step the H-score is related to a known reference value for each cell line expression level.

QUANTITATIVE OUTPUT VARIABLES
The output variables obtained from this protocol are: 

- Neg Nuclei (#): The number of negative nuclei

- 1+ Nuclei (#): The number of 1+ classified nuclei

- 2+ Nuclei (#): The number of 2+ classified nuclei

- 3+ Nuclei (#): The number of 3+ classified nuclei

-  Positive Percentage: The percentage of positive nuclei profiles

- H-score: (Percentage of 3+) x 3 + (Percentage of 2+) x 2 + (Percentage of 1+)

- Category: 0 if the cell line core is negative according to the reference H-score level for the PD-L1 negative cell line core, 1 if the cell line core is weak positive according to the reference H-score level for the PD-L1 weak positive cell line core, 2 if the cell line core is intermediate positive according to the reference H-score level for the PD-L1 intermediate positive cell line core, and 3 if the cell line core is strong positive according to the reference H-score level for the PD-L1 strong positive cell line core.

AUXILIARY APPS
There are no Auxiliary APPs available.

STAINING PROTOCOL
There is no staining protocol available.

ADDITIONAL INFORMATION
The APP utilizes the Visiopharm EngineTM and Viewer software modules, where EngineTM offers an execution platform to expand processing capability and speed of image analysis. The Visiopharm Viewer allows a fast review together with several types of image adjustment properties ex. outlining of regions, annotations and direct measures of distance, curve length, radius, etc. 
By adding the AuthorTM module the APP can be customized to fit other purposes. AuthorTM offers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required. For an optimized workflow the Tissuearray™ module is recommended as it facilitates easy and fast de-arraying of the cores.

REFERENCES
USER

The APP was developed for Horizon cell lines.
 

RUO
Figure 1
Figure 1
Zoomed in image of a strong postive cell line core.
Figure 2
Figure 2
The image from Figure 1, analyzed with the APP. Strong positive nuclei (3+) are labeled red, intermediate (2+) orange, weak (+1) yellow, and negative blue.
Figure 3
Figure 3
Zoomed in image of an intermediate positive cell line core.
Figure 4
Figure 4
The analyzed intermediate positive cell line core in Figure 3, demonstrating the detection of the varying staining intensity. Strong positive nuclei (3+) are labeled red, intermediate (2+) orange, weak (+1) yellow, and negative blue.
Figure 5
Figure 5
Zoomed in image of the weak positive cell line core.
Figure 6
Figure 6
The analyzed weak positive cell line core in Figure 5, demonstrating the detection of the varying staining intensity. Strong positive nuclei (3+) are labeled red, intermediate (2+) orange, weak (+1) yellow, and negative blue.
Figure 7
Figure 7
Negative cell line core.
Figure 8
Figure 8
The analyzed negative cell line core in Figure 7.