Increase in the presence of small vessels in poor prognosis FL identified as angiogenic sprouts using extended focal imaging. (A) CD31 was examined using TMA cores from extremes of survival diagnostic FL biopsies (< 5-year short-survival group and > 15-year long-survival group). Dot-plot chart shows significantly decreased interfollicular CD31⁺ mean vessel area (μm²) in the short-survival group. Each dot represents an FL patient (mean of triplicate cores) using the TMA. (B) Dot-plot chart shows significantly increased interfollicular CD31⁺ vascular structures measuring less than 30 μm² in the short-survival group. Each dot represents a single core (triplicate cores per patient) using the FL extremes of survival TMA. Statistical analysis was performed using a mixed-effects linear model (P values). The black bar represents the mean value for each experimental group. (C) Representative low power (original magnification ×20) images of CD31 interfollicular staining on TMA cores from the short-survival group (left-hand image) and the long-survival group (right-hand image). Note the increase in small, vascular structures measuring less than 30 μm² in the poor prognosis FL biopsy. The long-survival group exhibited larger mean vessel areas. Image acquisition: Olympus BX61 camera (Applied Imaging), 20×/0.5 numeric aperature (NA); images imported to Ariol Genetix Version 3.2.125 (Applied Imaging). (D) CD31 vascular structures present in the extremes of survival FL TMAs were examined using extended focal imaging (EFI). This technique builds a composite image of a focal series from 50-μm-thick whole sections that can be reconstructed with height lines in 3 dimensions (3-D) using image analysis software (process denoted by black arrows). This EFI analysis identified that the CD31⁺ vessels measuring less than 30 μm² in the short-survival FL group (C ●) were blind-ending sprouting processes. The white arrows indicate blind-ending vessels in the poor prognosis FL biopsies identified using EFI. Image acquisition: Olympus BX61 camera (Applied Imaging), 40×/0.75 NA; images constructed by CellF Version 24 (Olympus).