Elevated CD163⁺ macrophages correlate and are in close proximity with angiogenic sprouts in poor prognosis FL. (A) Dot-plot chart shows a positive correlation (Spearman rank coefficient, r = .43) of interfollicular CD163⁺ cells (rank density) with the numbers of CD31⁺ sprouting vessels (< 30 μm²). Representative images are shown on the right-hand side of CD31 and CD163 staining in the short-survival FL group using serial TMA sections. The merge image shows immunofluorescent analysis of these markers using a single section. Image acquisition: Olympus BX61 camera (Applied Imaging), 20×/0.5 NA; images imported to Ariol Genetix Version 3.2.125 (Applied Imaging). (B) Dot-plot chart shows significantly increased CD163⁺ macrophages in close proximity (within 200 μm) to a CD31⁺ sprouting vessel in the short-survival FL group compared with the long-survival group and reactive follicular hyperplasia controls. Each dot represents an average number of CD163⁺ macrophages from 15 analysis areas (200-μm area immediately surrounding a CD31⁺ sprouting vessel) for each patient sample. The red bar represents the mean value for each experimental group. Statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism Version 5.03 software; P values). Representative images are shown on the right-hand side of CD31 (labeled yellow using Ariol Genetix Version 3.2.125 analysis software [Applied Imaging]) and CD163 (labeled red) staining in the short- and long-survival FL TMA groups using linked serial TMA sections with Ariol image analysis software. Note the increase numbers of CD163⁺ macrophages within close proximity (200 μm, black arrow) to a CD31⁺ sprouting vessel (linked section, blue arrow) in the short-survival group (top images) compared with the long-survival group (bottom images).