Figure 4. (A) Original view and (B) view after applying the Positive Pixel Count algorithm v9 of digitized images of an ORO-stained frozen section at 40X magnification. The expert pathologist (AES) assessed global HS and MaS based on the original view, and the algorithm quantified the ORO stain percentage based on the stained slide pixel area. The ORO-stained lipid droplets (positive pixels) are in orange (A) in brightfield and in red (B) after the application of the Positive Pixel Count algorithm v9.
Considering the better linear correlation of the Raman signal with the TG results in the biochemical validation stage, compared to the Reflectance measurements, the Raman signal was used as the primary variable for predicting HS in the histopathological validation stage. The boxplots in Figure 5 show the distribution of fat content estimated by the Raman system, grouped by the degree of large droplet MaS (assessed by the expert pathologist), ORO stain percentage (estimated by the algorithm), and total steatosis percentage (evaluated by the expert pathologist).