Pathology
Following overnight fixation in 4% formaldehyde at room temperature,
resected fat tissue was subjected to a routine paraffin embedding
procedure. Sections 5 µm thick were obtained from paraffin blocks and
initially stained with hematoxylin-eosin using a Shandon Varistain
Gemini®(Shandon, Frankfurt) automated slide stainer. Additional
histochemical staining with periodic acid methenamine silver and
Masson’s trichrome was performed to assess morphology and the degree of
fibrosis in greater detail. All light microscopic evaluations were
conducted using an Olympus BX53® optical microscope. Measurements were
performed using digital photos captured using an Olympus SC50 CMOS®
camera and the drawing line tools in CellSens Entry Imaging
v.1.13®(Olympus Europa SE & Co. KG, Hamburg). Adipocyte diameter was
manually drawn and individually measured. In total, 350 adjacent
adipocytes were measured in each patient. Cells<35 μm were
excluded from analysis to avoid immature multilocular adipocytes.
Maximum adipocyte diameter and the presence of brown fat were noted
separately.
During microscopic examination the presence of fibrosis was observed in
some cases, primarily in the septae between fat lobules(i.e.,
perilobular fibrosis), around blood vessels(i.e., perivascular
fibrosis), and/or in the outermost Gerota’s fascia(Figure 1). Collagen
fibers were organized in bundles of variable thickness and some
exhibited spiculated infiltration to the periphery of the fat lobules,
isolating a few adipocytes from the remainder of the tissue. Moreover,
some cases exhibited pericellular fibrosis consisting of thinner
collagen fibrils surrounding adipocytes, localized in areas far from
fibrotic bundles(Figure 2). The presence or absence of these features
was recorded in each case. Additionally, maximum thickness of the
collagen at Gerota’s fascia and wall thickness of the thickest
vessel(from the endothelia to adventitia) in the resected fat were
measured, as described above. The number of vessels with a thick
wall(>100 µm) in a 1-cm2 area varied
according to patient and was scored as follows: 1: <3 vessels;
2: 3-5 vessels; 3: ≥6 vessels . Minimal inflammatory infiltrates that
contained a small number of lymphocytes and/or histiocytes existed in a
few cases and was also recorded.
Immunohistochemical staining with primary anti-CD31 antibody(Leica
Biosystems®; dilution,1:100; product code: NCL-L-CK7-560), an
endothelial marker, was performed to evaluate microvessel density in
perinephric fat. The Leica BOND-Max® automated staining platform (Leica
Microsystems, Wetzlar, Germany) and Bond Polymer Refine Detection
Kit®(Leica Biosystems, Newcastle Ltd., Newcastle Upon Tyne, UK; cat. no:
DS9800) were used for this purpose. In each case, 3 fields with the
highest number of microvessels were identified at low power. In these
areas mean capillary density (ACD) at 400× magnification was determined
and expressed as the number of CD31-positive capillaries per high power
field.
Specimens were qualified for histopathological examination in 55(88.7%)
of the 62 cases; 7 samples were excluded from the study due to poor
sampling or technical problems.