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.