Tissue T Cell Detection via Fluorescent CD45 Staining
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Solution Overview
Problem
Current methods are inadequate for determining the antigen specificity of T cells infiltrating tissues, especially when dealing with small cell numbers, as they fail to accurately detect and quantify antigen-specific T cells in tissue samples.
Innovation Solution
A method involving the simultaneous stimulation of isolated tissue cells with whole blood from the same person, using a fluorochrome-coupled CD45 antibody to differentiate between tissue and blood T cells, and analyzing cytokine induction via flow cytometry for improved detection and characterization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional stimulation methods are used on isolated T cells from tissue, then the stimulation process can be performed, but the detection sensitivity and signal strength are insufficient
Solution Approach 1:
The patent combines tissue T cells with whole blood from the same individual to create a mixed cell population for stimulation. This merging provides endogenous antigen-presenting cells and cytokines that enhance the stimulation signal, thereby improving detection sensitivity without requiring additional external components or complex stimulation systems.
Solution Approach 2:
The method uses the individual's own whole blood to provide antigen-presenting cells and cytokines necessary for T cell stimulation. This self-service approach eliminates the need for exogenous addition of complex antigen-presenting cell preparations or cytokine cocktails, simplifying the stimulation system while enhancing detection capability.
2Measurement precision
If tissue T cells are stained with fluorochrome-coupled CD45 antibody before mixing with blood cells, then tissue and blood T cells can be discriminated, but the procedure complexity increases
Solution Approach 1:
The patent applies fluorochrome-coupled CD45 antibody staining specifically to tissue T cells before mixing with whole blood cells. This localized staining creates a distinct fluorescent marker on tissue-derived T cells, enabling precise discrimination between tissue and blood T cell populations during flow cytometric analysis without requiring complex procedural modifications elsewhere in the workflow.
3Reliability
If whole blood is used for simultaneous stimulation of tissue and blood T cells, then the physiological milieu is optimized, but the ability to separately analyze tissue T cell response is compromised
Solution Approach 1:
The patent employs fluorochrome-coupled CD45 antibody staining that creates a fluorescent signal difference between tissue T cells (positive for tissue marker) and blood T cells (negative for tissue marker). This color/fluorescence change enables simultaneous stimulation in a physiological whole blood milieu while maintaining the ability to separately identify and quantify tissue T cell responses through flow cytometric detection of the fluorescent marker.
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 approach enables a more sensitive and quantitative analysis of antigen-specific T cells, facilitating diagnostic applications in infections, autoimmune diseases, and tumor diseases by allowing simultaneous quantification and analysis of tissue and blood T cells, enhancing prognostic assessment and treatment monitoring.
Implementation Method 1
The special feature of the new analysis method is that the cells isolated from tissue are stained with a fluorochrome-coupled CD45 antibody before they are mixed with the blood cells. As a result, in the final analysis, T cells from the blood and T cells from the tissue can be discriminated based on the absence or presence of this fluorescence signal
Implementation Method 2
The antigen-specific cells are quantified using flow cytometry based on the specific cytokine induction of T lymphocytes after stimulation
Data Source
AI summary
The invention relates to a method for the improved detection of antigen-specific, tissue-infiltrating immune cells, wherein the cells isolated from tissue are marked by means of a fluorochrome-coupled antibody, which is directed against antigens expressed on the cells, or by means of fluorochromes which are suitable for the coloring of living cells before they are mixed with heparinized whole blood.