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

Eosinophilic granulocytes are major effector cells in inflammation. Extrace llular deposition of toxic eosinophilic granule proteins (EGPs), but not th e presence of intact eosinophils, is crucial for their functional effect in situ. As even recent morphometric approaches to quantify the involvement o f eosinophils in inflammation have been only based on cell counting, we dev eloped a new method for the cell-independent quantification of EGPs by imag e analysis of immunostaining. Highly sensitive, automated immunohistochemis try was done on paraffin sections of inflammatory skin diseases with 4 diff erent primary antibodies against EGPs. Image analysis of immunostaining was performed by colour translation, linear combination and automated threshol ding. Using strictly standardized protocols, the assay was proven to be spe cific and accurate concerning segmentation in 8916 fields of 520 sections, well reproducible in repeated measurements and reliable over 16 weeks obser vation time. The method may be valuable for the cell-independent segmentati on of immunostaining in other applications as well.