Assessment of vascularity in histological sections: effects of methodology and value as an index of angiogenesis in breast tumours

The aims of this study were to (a) determine how the quantification of blood vessels in histological sections (vascularity) is affected by the methodology used and (b) assess the value of vascularity as an index of angiogenesis by comparing tumour and normal breast tissue. Archival specimens of breast, lung and oral carcinoma, oral dysplasia and normal breast tissue were used to test the effects of the following experimental variables on vascularity: pretreatment of the sections (enzymatic digestion, heating), endothelial markers (von Willebrand factor and CD31 antibodies), method of quantification (highest microvascular density, average microvascular density and microvascular volume) and interobserver variations. All the variables examined significantly affected the estimated vascularity; this depended on the type of tissue and method used. The pretreatment of the sections before staining was the most important variable, altering the vascularity ranking of the tumours. Vascularity in breast tumours was similar to that of the normal breast intralobular stroma, suggesting that an area of high microvascular density in the tumour does not necessarily represent tumour-induced angiogenesis. Contradictory results have been published regarding the value of vascularity as a tumour prognostic factor. Our results suggest that statistically significant differences in vascularity values are most likely to arise from failure to optimize the staining protocol and from the method used to assess vascularity.