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.