Elastin fiber morphometry
Elastin fibers in the alveolar walls were detected by VVG and orcein staining and images were captured as described above. Lengths of the elastin fibers were measured using ImageJ software analysis (National Institute of Health, April, 2019; ImageJ with 64-bit Java 1.8.0_172). Images from 10 randomly selected fields at 400x were captured. The images were first normalized for scale, the actual lengths of the elastin fibers were measured in micrometers (μm) in a blinded manner, and the curvatures of the elastin fibers along the walls of the alveoli were evaluated using freehand length measurement.41 The bundle of elastin fibers was measured as a single fiber unless the fibers were distinctly separated from each other. Elastin fibers often appear as single detached, fragmented or degraded fibers in COVID-19 patients. These fibers were counted separately to measure their lengths. All data were exported into an Excel file and the average fiber length in μm was determined. Each fiber was counted only once and no extra weighting was used for larger/wider fibers. Elastin fibers staining in the airways or surrounding bronchioles or pulmonary blood vessels were excluded.