Cell Identification via Blocking Polymer Exclusion
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Solution Overview
Problem
Current immunohistochemical techniques struggle to visualize multiple cell types simultaneously in a tissue section due to the lack of specific markers, leading to confounding cell types being misidentified, and existing methods are either tissue-destructive or insensitive, limiting the detection of cell markers in conventional histological sections.
Innovation Solution
A method involving a cocktail of antibody-like ligands that specifically bind to confounding cell markers, forming complexes and blocking polymers to exclude these cells, allowing for the visualization of particular cell types along with confounding cells using fluorochrome-labeled antibodies, enabling the detection of multiple cell populations in a single tissue section without tissue destruction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If multiple cell types are visualized simultaneously in one tissue section, then the spatial relationship between cell types can be studied, but confounding cell types may be misidentified due to overlapping markers
Solution Approach 1:
The method segments the identification process into multiple sequential steps: first visualizing cell types with unique markers, then selectively blocking their markers to prevent misidentification, and finally visualizing additional cell types. This segmentation allows simultaneous visualization of multiple cell types while maintaining identification accuracy through systematic exclusion of confounding cells.
Solution Approach 2:
The method performs preliminary blocking of confounding cell markers before visualizing the cell types of interest. By pre-blocked the markers on confounding cell types using antibody-like ligands and blocking polymers, the method ensures that subsequent visualization steps do not misidentify confounding cells, thus resolving the contradiction between simultaneous visualization and identification accuracy.
2Adaptability or versatility
If conventional IHC techniques are used to detect multiple cell markers, then tissue structure is preserved, but the number of detectable cell types is limited to 2-3 due to lack of appropriate antibody combinations
Solution Approach 1:
The method employs a universal detection system using fluorochrome-labeled secondary antibodies that can detect multiple different primary antibodies. This universal secondary antibody system eliminates the need for separate detection systems for each cell marker, allowing simultaneous detection of 5-8 different cell types in a single tissue section without increasing overall system complexity.
Solution Approach 2:
The method introduces blocking polymers as intermediary elements that selectively bind to and block confounding cell markers. These blocking polymers act as mediators that prevent false identification while allowing the primary detection system to function normally, thus expanding the number of detectable cell types without complicating the detection mechanism.
3Measurement precision
If tissue-destructive procedures are used to enable multiple staining, then detection sensitivity is improved, but the tissue sample is destroyed and cannot be further analyzed
Solution Approach 1:
The method replaces tissue-destructive mechanical procedures with a biochemical blocking approach using antibody-like ligands and blocking polymers. This substitution allows multiple staining and high detection sensitivity to be achieved through chemical blocking mechanisms rather than physical tissue destruction, preserving the integrity of the tissue sample for further analysis.
4Measurement precision
If simple blocking of confounding cells is attempted, then cell identification is improved, but the blocking method is either tissue-destructive or dependent on destruction of detection groups
Solution Approach 1:
The method changes the parameters of the blocking mechanism by using reversible, non-destructive blocking polymers that can be applied and removed without destroying tissue or detection groups. This parameter change from destructive to non-destructive blocking maintains identification accuracy while simplifying the methodology and enabling further analysis of the same tissue sample.
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
This method provides detailed information about the spatial relationship and visualization of multiple cell types and non-cellular structures in a single section, enhancing cell identification and allowing for further analysis of cell populations of interest, overcoming the limitations of existing techniques by enabling the detection of up to eight cell populations simultaneously.
Implementation Method 1
adding a first substrate preparation comprising a substrate for said first detection enzyme to the complex formed in step c) and incubating the resulting mixture under conditions that are favourable for formation of a detectable blocking polymer
Implementation Method 2
adding a first secondary antibody-like ligand preparation to the resulting mixture of step e), the first secondary antibody-like ligand being conjugated to a fluorochrome
Data Source
Figure 1a~1c
Figure 2
AI summary
The invention relates to a method of visualizing a plurality of cell types in a microscopic multicell tissue sample comprising cells of at least one particular cell type as well as cells of at least one confounding cell type, wherein cells of said at least one particular cell type and cells of said at least one confounding cell type both comprise a second cell marker and cells of the confounding cell type also comprise a first cell marker not present in cells of the at least one particular cell type, comprising the steps of exposing the cells of the sample to a first molecular detection means specifically binding to the first cell marker and having a detection enzyme; generating a blocking polymer within or around cells having the second cell marker by a reaction catalyzed by a detection enzyme on said first molecular detection means; exposing the cells of the sample to a second molecular detecting means comprising a fluorochrome, said second molecular detecting means specifically binding to said second cell marker; removing second molecular detection means that have not been bound to cells of the sample; and during the course of the method, visualizing said at least one particular cell type and said at least one confounding cell type by detecting fluorescence signals obtained from the fluorochrome of the second molecular detection means and, and by detecting the blocking polymer that has been formed around said at least one confounding cell type.