All iris tissue samples were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin (HE) to evaluate the following histologic features: (1) the presence of lymphocyte infiltration, (2) the presence of plasma cells, (3) the presence of macrophages, and (4) fibrosis of iris stroma. A minimum of four sections was obtained from each specimen. The sections were examined using a light microscopy at a ×400 magnification by a single masked pathologist who was not aware of the diagnosis. A high power field (HPF) with highest cellularity was chosen for analysis. Because of the small size of the specimens no difficulties were encountered when choosing a representative HPF to assess the inflammatory status of the specimen. A semiquantitative scoring system was used with a scale ranging from 0 to 4 depending on the quantity of positively stained cells present. A score of 1 represented mild infiltration (1–3 cells in a HPF), while a score of 4 represented infiltration by numerous inflammatory cells (>20 cells in a HPF). In specimens with a detectable inflammatory infiltrate, additional immunostaining was performed.
The primary antibodies used included a monoclonal rabbit anti-CD4 for T helper cells (SP35, 104R-16, 1:50; Cell Marque, Rocklin, CA, USA), monoclonal mouse anti-CD8 for cytotoxic T cells (M7103, 1:50; Dako, Glostrup, Denmark), monoclonal mouse anti-CD20 for B lymphocytes (M755, 1:800; Dako), monoclonal mouse CD68 for macrophages (NCL-CD68, 1:1600; Novocastra via Leica Biosystems, Nussloch, Germany), and mouse anti-CD138 (MCA 68-1H, 1:500; Serotec, Kidlington, UK) to identify the presence of plasma cells. A horseradish peroxidase (HRP) technique was used in an automated immunostainer. Appropriate antibody isotype control has been performed using tonsil tissue for anti-CD4, CD8, CD 20, and CD 68, and bone marrow for anti-CD 138. Immunohistochemical sections were examined under a light microscope at ×400 magnification.