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.