Quantification of eosinophilic granule protein deposition in biopsies of inflammatory skin diseases by automated image analysis of highly sensitive immunostaining

Abstract

Eosinophilic granulocytes are major effector cells in inflammation. Extracellular deposition of toxic eosinophilic granule proteins (EGPs), but not the presence of intact eosinophils, is crucial for their functional effect in situ. As even recent morphometric approaches to quantify the involvement of eosinophils in inflammation have been only based on cell counting, we developed a new method for the cell-independent quantification of EGPs by image analysis of immunostaining. Highly sensitive, automated immunohistochemistry was done on paraffin sections of inflammatory skin diseases with 4 different primary antibodies against EGPs. Image analysis of immunostaining was performed by colour translation, linear combination and automated thresholding. Using strictly standardized protocols, the assay was proven to be specific and accurate concerning segmentation in 8916 fields of 520 sections, well reproducible in repeated measurements and reliable over 16 weeks observation time. The method may be valuable for the cell-independent segmentation of immunostaining in other applications as well.