After obtaining approval from the Mayo Clinic Institutional Review Board, we reviewed the records of two patients with choroidal melanomas who had enucleations at Mayo Clinic in Rochester, MN. Our study complied with the Declaration of Helsinki.
Both patients had a complete ocular examination, which included Snellen visual acuity, slit-lamp biomicroscopy, intraocular pressure, and funduscopy. Furthermore, optical coherence tomography (OCT), color fundus photographs, and autofluorescence photographs also were obtained. Fundus autofluorescence (FAF) photographs were attained with the Heidelberg confocal scanning laser ophthalmoscope system (Hiedelberg Retina Angiograph [HRA]; Hiedelberg Engineering, Dossenheim, Germany). In this machine, autofluorescence is excited by using argon blue wavelength (488 nm). We examined the color fundus and autofluorescence photographs for the presence of orange pigment and autofluorescence, respectively.
The enucleated eyes were fixed for a minimum of 48 hours in buffered formalin at 10%. Following fixation, these eyes were examined, photographed, and sectioned following guidelines for examination of enucleated eyes with uveal melanoma.
20 Sections of the eyes were paraffin-embedded and submitted to histologic preparation and staining with hematoxylin-eosin (H&E) and periodic acid–Schiff (PAS) for subsequent microscopic examination.
Additional sections were examined from areas identified as orange pigment–rich by comparison with the autofluorescence photographs. Using fluorescent microscopy, a few unstained sections were evaluated for the location of areas with higher concentration of autofluorescent lipofuscin. Selected tissue sections were stained with immunohistochemistry (
Table 1). Antibodies against CD68 and CD163 were used to identify macrophages. Antibodies against S-100 and cytokeratins: keratin 7, CAM 5.2 (keratins 7 and 8), AE1/AE3 (keratins 1–8, 10, 14–16, and 19), and OSCAR Keratin (pan-keratin cocktail) were used to immunophenotype the RPE cells.
Unstained tissue sections with higher concentration of autofluorescent lipofuscin were selected and marked to be evaluated by electron microscopy; 10-μm paraffin-embedded tissue sections were placed onto glass slides. The unstained sections were deparaffinized by placing the slide into warm xylene for 60 minutes. The slide then was transferred to absolute ethanol at 60°C for 30 minutes followed by a decreasing concentration of ethanol to 60%. The slide then was fixed in Trump's fixative (1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2) overnight.
21 The slide then was rinsed for 30 minutes in three changes of 0.1 M phosphate buffer, pH 7.2, followed by a 1-hour postfix in phosphate-buffered 1% osmium tetroxide (OsO4). After rinsing in three changes of distilled water for 30 minutes, the tissue was en bloc stained with 2% uranyl acetate for 30 minutes at 600C. After en bloc staining, the tissue was rinsed in three changes of distilled water, dehydrated in progressive concentrations of ethanol and 100% propylene oxide, and embedded in Spurr's resin.
22 Thin (90 nm) sections were cut on a Leica UC6 ultramicrotome (Leica, Wetzlar, Germany), placed on 200 mesh copper grids, and stained with lead citrate. Micrographs were taken on a JEOL 1400 plus transmission electron microscope (JEOL USA, Inc., Waterford, VA) operating at 80 KV.