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Updated: Aug 19, 2026
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
Analysis of the reliability of manual and automated immunohistochemical staining procedures. A pilot study
Stefan Biesterfeld1, Helen Louise Kraus, Thorsten Reineke
1Department of Pathology, Technical University of Aachen, Pauwelsstrasse 30, 52057 Aachen, Federal Republic of Germany. biesterfeld@pathologie-re.de
Objective:
To study the variation in the number of stained cells and staining intensity comparing 2 immunostainers and manual staining for estrogen receptor (ER) expression in breast carcinoma.
Study Design:
In 5 cases, 15 consecutive paraffin sections were investigated after simultaneous immunohistochemical ER staining. The slides were evaluated using a CM-2 TV image analysis system (Hund, Wetzlar, Germany). One viewing field, identified around a histologic structure present on all 15 sections, was analyzed. The percentage of immunoreactive cells (PP), mean grey values of the immunopositive (GVpos.) and immunonegative nuclei (GVneg.), and immunohistochemical staining intensity (SI, defined as GVneg.-GVpos.) were calculated.
Results:
The mean PP values were higher for immunostainers A (70.2%) and B (53.8%) than for manual staining (40.8%). The results were significantly different comparing the 2 immunostainers (P = .0143) or immunostainer A and manual staining (P < .0001). Also, the mean SI values were higher for immunostainers A (24.5 +/- 2.8% [CV]) and B (18.5 +/- 31.1%) than for manual staining (10.8 +/- 33.8%). These differences revealed statistical significance comparing the immunostainers with manual staining (.0001 < P = .0048).
Conclusion:
Our results underline the higher staining quality using immunostainers in comparison with manual staining.
