Quantification of collagen and measurement of collagen:elastin ratio in the lungs of COVID-19 patients.
Masson’s Trichrome stained lung sections were imaged as described above, and images were analyzed by ImageJ software. For the measurement of collagen density, 10 random fields were selected from each lung section. The collagen staining was processed by selecting Masson’s Trichrome specific staining using color deconvolution plugin software in ImageJ that separates red, blue, and green components from the image. Thresholds for collagen staining were established for each slide by enhancing the contrast to a point at which the collagen staining was easily identified by blue staining and the density of the collagen was measured over total lung area from the image.42 The collagen was measured as percent collagen density in total lung area of the image. No weighting was performed for the intensity of blue staining. Further, adjacent serial lung sections were stained with Masson’s Trichrome and orcein for quantitative measurement of collagen:elastin ratio as a fold change after both stains were normalized to the total lung area in the image captured in the same areas of the sections.