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Updated: Aug 8, 2026
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
Quantitative immunohistochemistry using the CAS 200/486 image analysis system in invasive breast carcinoma: a
S V Makkink-Nombrado1, J P Baak, L Schuurmans
1Department of Pathology, Free University Hospital, Amsterdam, The Netherlands.
Insights
Reproducibility in quantitative immunohistochemistry (IHC) analysis of breast cancer biomarkers like Estrogen Receptor (ER) and Ki67 is excellent within observers but lower between them. Strict sampling protocols are essential for reliable IHC results.
Area of Science:
- Pathology
- Oncology
- Biomedical Imaging
Background:
- Quantitative immunohistochemistry (IHC) is crucial for breast cancer diagnosis and treatment.
- Ensuring reproducibility in IHC analyses is vital for accurate patient management.
Purpose of the Study:
- To evaluate the intra- and inter-observer reproducibility of quantitative IHC analyses for key breast cancer biomarkers.
- To identify factors contributing to variability in IHC assessments.
- To compare quantitative digital image analysis with qualitative visual assessment.
Main Methods:
- Quantitative IHC analysis of Estrogen Receptor (ER), Progesterone Receptor (PR), Ki67, HER-2/neu (c-erbB-2), and cathepsin D (CD) using the CAS 200/486 image analyzer.
- Qualitative IHC analysis of Epidermal Growth Factor Receptor (EGF-R).
- Duplicate blind assessments by the same observer and assessments by three different operators.
Main Results:
- Excellent intra-observer reproducibility was observed for all quantitative IHC markers.
- Lower inter-observer reproducibility was found, with agreement ranging from 56% to 79% for quantitative markers.
- Variation in sampling fields within a section was identified as the major cause of inter-observer variability.
Conclusions:
- While intra-observer agreement is high, inter-observer variability in quantitative IHC can lead to systematic differences.
- A strict sampling protocol for fields of vision is mandatory to achieve reproducible quantitative IHC results.
- Convenience sampling is insufficient for ensuring adequate and reproducible IHC analysis.
Abstract:
We evaluated the intra- and inter-observer reproducibility of quantitative immunohistochemical (IHC) analyses using the Cell Analysis Systems (CAS) 200/486 image analyzer of Estrogen Receptor (ER), Progesterone Receptor (PR), proliferation-associated nuclear protein (Ki67), HER-2/neu (c-erbB-2) protein over-expression and cathepsin D (CD) in 20 randomly-selected invasive breast carcinomas. Qualitative analysis of IHC Epidermal Growth Factor Receptor (EGF-R) was also assessed in this study for comparative purposes. Duplicate blind assessments by the same observer showed excellent correlations for all quantitative IHC features (P < 0.001; P = 0.004 for neu). However, the immuno-quantitative analyses results between the 3 different operators showed lower correlation coefficient values, thus being less reproducible. This resulted in systematic differences and bias between the observers. This was also clear from the overall agreement between the 3 observers which was 70% for ER, 70% for PR, 56% for Ki67, 79% for c-erbB-2 and 75% for CD. The qualitative visual assessments of EGF-R, expressed as either positive or negative, showed a 75% agreement between observers and 85% intra-observer agreement (comparable to quantitative digital image processing results). The same results were obtained with kappa statistics. A further analysis of the factors causing the lack of reproducibility was performed. For quantitative IHC, segmentation of stored and retrieved digitized images was quite reproducible between and within well-trained observers. However, variation between different fields of vision of one and the same section showed large variations for most cases. Therefore, differences in sampling of fields within a section appeared to be the major cause of lack of reproducibility between observers, although segmentation differences still added slightly to the inter-observer variations. Accordingly, a strict sampling protocol of fields of vision is mandatory to obtain reproducible quantitative IHC results. It is clear from the present study that so-called random (but in fact, at convenience) selection of fields of vision for measurement is not a sufficient guarantee of adequacy of the sampling.
