6th ESACP Congress, Heidelberg, April 7-11, 1999

A080
QUANTIFICATION OF EOSINOPHILIC GRANULE PROTEINS IN SKIN BIOPSIES BY IMAGE ANALYSIS OF IMMUNOSTAINING USING COLOR TRANSLATION, LINEAR COMBINATION AND AUTOMATED THRESHOLDING
Kiehl P, Falkenberg K, Vogelbruch M, Kapp A

Department of Dermatology and Allergology, Hannover Medical University, Germany

BACKGROUND: Eosinophilic granulocytes are major effector cells in allergic inflammation. Morphometric approaches to quantify their involvement in inflammation are mainly based on cell counting. However, extracellular tissue deposition of toxic eosinophilic granule proteins (EGPs),but not only the presence of intact eosinophils is crucial for their functional effect in situ. OBJECTIVE: To develop a method of quantitating extra- and intracellular EGPs in skin biopsies by image analysis of immunostaining. METHODS: High sensitive immunohistochemistry was done on paraffin sections of eosinophil-rich inflammatory dermatoses with 4 different primary antibodies against EGPs (MBP, EG1, EG2, EPO) using an automated immunostainer and newfuchsin as chromogen without counterstaining. Image analysis was performed with a Olympus-BH2-microscope (SplanApo20x-objektive), a Sony DXC950 video camera on a 0.5 reduction-tube and the software analySIS 3.0 (SIS, Münster, Germany). Based on methods by RUIFROK1997 and SMOLLE1996 six to eight fields per section were analysed as follows: shading-correction; translation of the true color image to 8-bit-grayscale in the inverted red channel (Rinv) of RGB and the saturation channel (S) of HSI; linear combination of Rinv and S; automated thresholding based on t-Test statistics; measurements of the area fraction of immunostaining as main parameter. RESULTS AND CONCLUSIONS: Out of different approaches, this method was most accurate in detecting red immunostaining specifically and reproducibly in 60 repeated measurements on different days (CV < 5%).Unwanted segmentation of brown endogenous melanin pigment could be avoided. The method may be of value for the segmentation of immunostaining not restricted to cells in other problems as well.