Multiplexed Immunohistochemical Staining for Tissue Profiling
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Solution Overview
Problem
Current methods for high-dimensional immunohistochemical analyses of tumor tissues are limited by the availability of tumor tissue, reliance on proprietary fluorescent probes, requirement for costly equipment and reagents, and inability to perform comprehensive analysis of immune cell populations, which hampers the identification of predictive markers for immunotherapy response.
Innovation Solution
The Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS) method, which allows iterative cycles of staining and destaining on formalin-fixed paraffin-embedded tissues using chromogenic substrates, enabling the characterization of multiple immune markers on a single slide without altering tissue antigenicity and allowing for prolonged storage, combined with automated digital landscaping for complex analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If commercial multiplexed immunostaining methods are used to allow high dimensional analysis of complex immune cell populations, then the analysis capability is improved, but the cost of equipment, materials, and reagents increases significantly
Solution Approach 1:
The patent uses standard chromogenic substrates (DAB, AEC, FAST) that are inexpensive and can be discarded after a single staining cycle, replacing expensive proprietary fluorescent probes. Each substrate is used once then discarded, but the low cost allows multiple staining cycles on the same tissue section, achieving high-dimensional analysis without substantial equipment or reagent investment
Solution Approach 2:
The patent employs a universal chromogenic substrate system where multiple different substrates (DAB, AEC, FAST) can be used sequentially on the same tissue section to detect multiple markers. This multi-functional approach allows a single tissue sample to be analyzed for numerous immune cell populations using standard, inexpensive reagents rather than requiring specialized expensive equipment and proprietary probes
2Reliability
If frozen material is used for multiplexed immunostaining to preserve tissue antigenicity, then the antigenicity is maintained, but the tissue availability is reduced and storage requirements become more complex
Solution Approach 1:
The patent changes the chemical parameters of the staining process by using alcohol-soluble chromogenic substrates (AEC, FAST) that can be completely removed by ethanol washing. This parameter change allows the use of standard formalin-fixed paraffin-embedded tissues without freezing, maintaining tissue availability while preserving antigenicity through optimized antigen retrieval protocols that work with FFPE samples
3Adaptability or versatility
If multiple markers are stained simultaneously on a single tissue slide to enable comprehensive immune cell population analysis, then the analysis comprehensiveness is improved, but the tissue availability is depleted
Solution Approach 1:
The patent segments the staining process into multiple sequential cycles, each detecting one or a few markers. After each staining cycle, the chromogenic substrate is completely removed by ethanol washing, erasing the previous staining pattern. This segmentation allows the same tissue section to be stained for multiple different markers in sequence, achieving comprehensive high-dimensional analysis without consuming additional tissue samples
Solution Approach 2:
The patent discards the chromogenic substrate staining product after image acquisition by washing with ethanol, which completely removes alcohol-soluble substrates like AEC and FAST. This discarding step recovers the tissue section in an antigen-positive state, allowing subsequent staining cycles for other markers. The substrate is discarded but the tissue is recovered for repeated use
4Reliability
If chromogenic substrates are used for iterative staining cycles to enable sample-sparing analysis, then the tissue integrity is preserved, but the chromogen removal efficiency must be optimized to prevent cross-contamination
Solution Approach 1:
The patent changes the chemical parameter of the chromogenic substrates by selecting alcohol-soluble variants (AEC, FAST) instead of water-soluble ones. This parameter change enables complete removal of the chromogen through ethanol washing, achieving effective cross-contamination prevention while preserving tissue integrity. The alcohol solubility parameter allows simple yet thorough substrate removal without complex procedures
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables comprehensive, sample-sparing, and cost-effective high-dimensional analysis of tumor tissues, facilitating the identification of predictive biomarkers and monitoring of immune responses to immunotherapy, while preserving tissue integrity and allowing for prolonged storage of slides.
Implementation Method 1
Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole (AEC), combined with a rapid non-destructive method for antibody-antigen dissociation, the method allows to erase the results of a single immunohistochemical stain while preserving tissue antigenicity
Data Source
Figure 1A~1B
Figure 2A~2L
Figure 3A
AI summary
The present invention relates to methods and compositions for sequential multidimensional immunohistochemical analyses of tissues.