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Physical basis of the ‘magnification rule’ for standardized Immunohistochemical scoring of HER2 in breast and gastric cancer

Overview of attention for article published in Diagnostic Pathology, March 2018
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Physical basis of the ‘magnification rule’ for standardized Immunohistochemical scoring of HER2 in breast and gastric cancer
Published in
Diagnostic Pathology, March 2018
DOI 10.1186/s13000-018-0696-x
Pubmed ID

Andreas H. Scheel, Frédérique Penault-Llorca, Wedad Hanna, Gustavo Baretton, Peter Middel, Judith Burchhardt, Manfred Hofmann, Bharat Jasani, Josef Rüschoff


Detection of HER2/neu receptor overexpression and/or amplification is a prerequisite for efficient anti-HER2 treatment of breast and gastric carcinomas. Immunohistochemistry (IHC) of the HER2 protein is the most common screening test, thus precise and reproducible IHC-scoring is of utmost importance. Interobserver variance still is a problem; in particular in gastric carcinomas the reliable differentiation of IHC scores 2+ and 1+ is challenging. Herein we describe the physical basis of what we called the 'magnification rule': Different microscope objectives are employed to reproducibly subdivide the continuous spectrum of IHC staining intensities into distinct categories (1+, 2+, 3+). HER2-IHC was performed on 120 breast cancer biopsy specimens (n = 40 per category). Width and color-intensity of membranous DAB chromogen precipitates were measured by whole-slide scanning and digital morphometry. Image-analysis data were related to semi-quantitative manual scoring according to the magnification rule and to the optical properties of the employed microscope objectives. The semi-quantitative manual HER2-IHC scores are correlated to color-intensity measured by image-analysis and to the width of DAB-precipitates. The mean widths ±standard deviations of precipitates were: IHC-score 1+, 0.64 ± 0.1 μm; score 2+, 1.0 ± 0.23 μm; score 3+, 2.14 ± 0.4 μm. The width of precipitates per category matched the optical resolution of the employed microscope objective lenses: Approximately 0.4 μm (40×), 1.0 μm (10×) and 2.0 μm (5×). Perceived intensity, width of the DAB chromogen precipitate, and absolute color-intensity determined by image-analysis are linked. These interrelations form the physical basis of the 'magnification rule': 2+ precipitates are too narrow to be observed with 5× microscope objectives, 1+ precipitates are too narrow for 10× objectives. Thus, the rule uses the optical resolution windows of standard diagnostic microscope objectives to derive the width of the DAB-precipitates. The width is in turn correlated with color-intensity. Hereby, the more or less subjective estimation of IHC scores based only on the staining-intensity is replaced by a quasi-morphometric measurement. The principle seems universally applicable to immunohistochemical stainings of membrane-bound biomarkers that require an intensity-dependent scoring.

Mendeley readers

The data shown below were compiled from readership statistics for 17 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 17 100%

Demographic breakdown

Readers by professional status Count As %
Researcher 3 18%
Student > Postgraduate 3 18%
Student > Doctoral Student 2 12%
Student > Bachelor 1 6%
Professor 1 6%
Other 4 24%
Unknown 3 18%
Readers by discipline Count As %
Medicine and Dentistry 7 41%
Engineering 2 12%
Computer Science 2 12%
Unspecified 1 6%
Chemistry 1 6%
Other 1 6%
Unknown 3 18%

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