Figure 1. Flow chart of the present study. (a) The specimens had
tumours, and the stained frozen section was physically close to the
tumour. (b) The SD of voltage signals in either channel was greater than
80 µV.
Stage I: biochemical
validation
In the first stage, we studied 25 snap-frozen human liver specimens
(weighing between 1.3 mg and 58.1 mg): 24 from remnants of pre- and
post-LT wedge biopsies and one from a discarded donor liver at Leiden
University Medical Center (LUMC) in August 2022. The specimens were
thawed at room temperature and scanned using the Raman system under
ambient room light. The liver specimens were refrozen at -80 degrees
Celsius and thawed again for TG quantification. Given that the
penetration depth of the incident laser beam was at least
1mm,26 only specimens heavier than 10 mg (thicker than
1 mm) were included to avoid the insufficiency to quantify TG of
low-mass specimens27,28. The fat content derived from
the analyses using the Raman and reflectance channels was then compared
with the TG quantification results to assess the Raman system’s accuracy
in steatosis estimation.
Stage II:
histopathological
validation
In the second stage, at Vanderbilt University Medical Center (VUMC) in
November 2022, 70 snap-frozen human liver specimens weighing between
0.24 g and 2.58 g were obtained through the Cooperative Human Tissue
Network. These liver specimens were split on dry ice: one half was
thawed in cold water and scanned under ambient light using the
investigated Raman system, while the other half was cryo-sliced and
stained with Oil Red O (ORO) for histological analysis. The Raman system
user (HG) and the expert pathologist (AES) performed the Raman and
histopathological assessments independently, blinded to the results of
each other assessment. Four liver specimens were excluded due to
proximity to tumours or due to Raman data collection issues. The Raman
results were compared to expert pathologist evaluations of steatosis.