Related Experiment Video
Updated: Apr 30, 2026
Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
Published on: January 21, 2019
Sequential immunohistochemistry: a promising new tool for the pathology laboratory
Michiel van den Brand1, Brigiet M Hoevenaars, Jessica H M Sigmans
1Department of Pathology, Radboud University Medical Centre, Nijmegen, The Netherlands.
Insights
Sequential immunohistochemistry (S-IHC) enables studying multiple proteins in one tissue slide, overcoming limitations of current methods. This technique revealed discrepancies in lymphoma diagnoses, highlighting its diagnostic value.
Area of Science:
- Histopathology
- Immunohistochemistry
- Oncology
Background:
- Conventional immunohistochemistry (IHC) has limitations for multi-protein analysis in single tissue sections.
- Sequential IHC (S-IHC) offers a novel solution for studying numerous proteins within one section with minimal optimization.
Purpose of the Study:
- To introduce and validate sequential immunohistochemistry (S-IHC) as an improved method for multi-protein analysis.
- To assess the utility of S-IHC in characterizing lymphomas.
Main Methods:
- Tissue sections were stained sequentially with multiple antibodies, with intermediate scanning and elution of reagents.
- Digital images were overlaid to enable simultaneous assessment of multiple protein expressions.
- S-IHC was applied to nine nodular lymphocyte-predominant Hodgkin lymphomas (NLPHLs) and ten T-cell-rich/histiocyte-rich diffuse large B-cell lymphomas (T/HRBCLs).
Main Results:
- Expression of cyclin D1, CD20, and CD68 was analyzed in lymphoma subtypes.
- Cyclin D1 was detected in 44% of NLPHLs and 60% of T/HRBCLs.
- S-IHC identified discrepancies in 42% of cases compared to single IHC, underscoring challenges in distinguishing tumor cells from histiocytes.
Conclusions:
- S-IHC is a valuable technique for research and diagnostics, enabling comprehensive protein expression analysis in single tissue sections.
- The method preserves tissue architecture, facilitating detailed phenotypic characterization of individual cells.
- S-IHC demonstrates significant added value in complex cases like lymphomas where cell morphology can be ambiguous.
Aims:
Current immunohistochemical methods to study the expression of multiple proteins in a single tissue section suffer from several limitations. In this article, we report on sequential immunohistochemistry (S-IHC), a novel, easy method that allows the study of numerous proteins in a single tissue section, while requiring very limited optimization.
Methods And Results:
In S-IHC, a tissue section is stained for multiple antibodies, with intermediate scanning of the section and elution of chromogen and antibodies. Overlays are made of the digital images, allowing assessment of multiple proteins in the same tissue section. We used S-IHC to study nine nodular lymphocyte-predominant Hodgkin lymphomas (NLPHLs) and 10 T-cell-rich and histiocyte-rich diffuse large B-cell lymphomas (T/HRBCLs) for expression of cyclin D1, CD20, and CD68. We observed cyclin D1 expression in single tumour cells in 44% of NLPHLs and 60% of T/HRBCLs. Comparison of S-IHC with classic single immunohistochemical staining revealed discrepancies in eight cases (42%), demonstrating the difficulty of differentiating tumour cells from histiocytes on morphological grounds, and stressing the additional value of S-IHC.
Conclusions:
For research and diagnostic purposes, S-IHC is a promising technique that assesses the expression of numerous proteins in single tissue sections with complete architectural information, allowing phenotypic characterization of single cells.
