Figure 2.
Correlation analysis between the pathologist’s IHC scores for BCL2 staining and the quantified values obtained by image analysis. (A) A digital slide image is shown for the BCL2 IHC each for score 3+ to 0 (top). The bottom figure is the corresponding artificial image produced with the positive pixel count algorithm (PPC). The positive brown DAB (3,3′-diaminobenzidine) staining is colored according to the level of pixel intensity with red (strong), orange (medium), yellow (weak), and blue (negative, nuclear counter stain). The thresholds for the weak, medium, and strong intensities of staining were set as defaults. Because BCL2-positive reactive T cells are always admixed in the tumor areas, these small T cells are manually selected and separated from the large tumor cells in each ROI, and the total sum of these individual areas are evaluated, respectively. Clusters of large tumor cells and individual small T cells are manually delineated by the gray lines and light blue circles, respectively. (B) In each ROI, the number of strong-positive pixels was automatically counted, and the percentage of total number of positive-staining pixels was plotted. The box and plots show the distribution of the percentage. The median values of the score of 3+ group showed a significantly higher than the score of 2+ group (P < .0001, Mann-Whitney U test for pairwise comparison with Bonferroni correction), whereas no significant difference was observed among score 0 to 2+ groups. The horizontal line within the box indicates the median value, and the lower and upper portions indicate the 25% and 75% interquartile range, respectively. The error bars represent the 5% and 95% quantiles. (C) To evaluate the staining intensity of the tumor cells, the average intensity values of the tumor cells are compared after normalization to the T cells. The intensity ratio (tumor/normal T cell) was plotted; a significant positive trend was observed for the tumor staining intensity levels with the IHC score groups (P < .0001, respectively, Mann-Whitney U test for pairwise comparison with Bonferroni correction). *Statistically significant; NS, not significant.

Correlation analysis between the pathologist’s IHC scores for BCL2 staining and the quantified values obtained by image analysis. (A) A digital slide image is shown for the BCL2 IHC each for score 3+ to 0 (top). The bottom figure is the corresponding artificial image produced with the positive pixel count algorithm (PPC). The positive brown DAB (3,3′-diaminobenzidine) staining is colored according to the level of pixel intensity with red (strong), orange (medium), yellow (weak), and blue (negative, nuclear counter stain). The thresholds for the weak, medium, and strong intensities of staining were set as defaults. Because BCL2-positive reactive T cells are always admixed in the tumor areas, these small T cells are manually selected and separated from the large tumor cells in each ROI, and the total sum of these individual areas are evaluated, respectively. Clusters of large tumor cells and individual small T cells are manually delineated by the gray lines and light blue circles, respectively. (B) In each ROI, the number of strong-positive pixels was automatically counted, and the percentage of total number of positive-staining pixels was plotted. The box and plots show the distribution of the percentage. The median values of the score of 3+ group showed a significantly higher than the score of 2+ group (P < .0001, Mann-Whitney U test for pairwise comparison with Bonferroni correction), whereas no significant difference was observed among score 0 to 2+ groups. The horizontal line within the box indicates the median value, and the lower and upper portions indicate the 25% and 75% interquartile range, respectively. The error bars represent the 5% and 95% quantiles. (C) To evaluate the staining intensity of the tumor cells, the average intensity values of the tumor cells are compared after normalization to the T cells. The intensity ratio (tumor/normal T cell) was plotted; a significant positive trend was observed for the tumor staining intensity levels with the IHC score groups (P < .0001, respectively, Mann-Whitney U test for pairwise comparison with Bonferroni correction). *Statistically significant; NS, not significant.

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