(A) A ‘screen grab’ image demonstrating the process of quantifying a radiographic injection study of the gracilis muscle and overlying fasciocutaneous tissues.

Image was opened in Image J® and ‘initialized’ in the LVAP ‘Lymphatic Vessel Analysis Protocol’ (LVAP) plug-in. The image was then overlayed with two grids (see methods) to allow systematic quantification of vessel density. A mouse click is made at the commencement of a vessel as it crosses a grid line and a second click on opposite side of the same vessel. The LVAP program then measures the intervening distance as vessel width for the given vessel, and all widths for the viewing screen (representing one third of the specimen) are averaged and collated. (B) Graphical representation of the quantified average vessel width in both the muscle and skin specimens, in each one third of the flap.