ImmunoMembrane: a publicly available web application for digital image analysis of HER2 immunohistochemistry

Histopathology. 2012 Apr;60(5):758-67. doi: 10.1111/j.1365-2559.2011.04142.x. Epub 2012 Feb 1.


Aims: Assessment of the human epidermal growth factor receptor 2 (HER2) with immunohistochemistry (IHC) is routine practice in clinical pathology laboratories. Visual classification of the staining reaction (usually into 0/1+, 2+ or 3+) is subjective and prone to significant inter- and intra-observer variation. In this study, we describe ImmunoMembrane, an easy-to-use HER2 IHC analysis software, which is freely available as a web application, requiring no download or installation.

Methods and results: ImmunoMembrane uses colour deconvolution for stain separation and a customized algorithm for cell membrane segmentation. A quantitative score (IM-score, 0-20 points) is generated according to the membrane staining intensity and completeness. Specimens are classified into 0/1+, 2+ or 3+ based on IM-score cut-offs defined using a training set. The classification and membrane segmentation are presented as a pseudo-coloured overlay image. With a validation set (144 HercepTest(®) -stained whole tissue sections), ImmunoMembrane matched well with the pathologist's visual classification (weighted kappa κ(w) =0.80), as well as fluorescence in-situ hybridization (FISH) (IHC disagreement 3.5%, n=144) and chromogenic in-situ hybridization (CISH) (IHC disagreement 2.8%, n=144).

Conclusions: We anticipate that publicly available web applications, such as ImmunoMembrane, will accelerate the adoption of automated image analysis in clinical diagnostics of HER2 IHC. ImmunoMembrane is freely accessible at:

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Biomarkers, Tumor / metabolism
  • Breast Neoplasms / diagnosis*
  • Breast Neoplasms / metabolism
  • Chromogenic Compounds
  • DNA, Neoplasm / analysis
  • Female
  • Genetic Markers
  • Humans
  • Image Processing, Computer-Assisted*
  • Immunohistochemistry / methods*
  • In Situ Hybridization, Fluorescence
  • Receptor, ErbB-2 / genetics
  • Receptor, ErbB-2 / metabolism*
  • Reproducibility of Results
  • Software
  • Tissue Array Analysis


  • Biomarkers, Tumor
  • Chromogenic Compounds
  • DNA, Neoplasm
  • Genetic Markers
  • ERBB2 protein, human
  • Receptor, ErbB-2