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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A080
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.
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