PD-L1 Detection via EpCAM Capture Screen and Multicolor Fluorescence
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Solution Overview
Problem
Current methods for detecting PD-L1 on tumor cells, particularly circulating tumor cells, are invasive, inaccurate, and subject to high variability due to complex background cells and expert-dependent interpretation.
Innovation Solution
A novel method utilizing a capture screen with EpCAM antibodies to specifically capture tumor cells from body fluids, followed by incubation with PD-L1 antibodies and multicolor imaging analysis using DAPI, CK, and CD45 fluorescent antibodies to accurately identify CTCs with PD-L1 expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If immunohistochemical methods are used to detect PD-L1 on tumor tissues, then detection can be performed on tumor samples, but the results are closely related to pathologist experience and lack stability
Solution Approach 1:
The patent replaces manual immunohistochemical staining and expert visual interpretation with automated flow cytometry technology. The system uses automated cell capture, staining, and analysis equipment to objectively detect PD-L1 expression, eliminating the subjectivity and experience-dependency of pathologist interpretation while maintaining detection accuracy.
Solution Approach 2:
The patent employs automated flow cytometry systems that perform cell capture, antibody staining, washing, and data analysis automatically without manual intervention. The system self-calibrates and processes samples consistently, ensuring reproducible results across different operators and laboratories, thereby improving result stability.
2Quantity of substance
If whole blood is treated with red cell lysing solution to separate nucleated cells for PD-L1 detection, then nucleated cells can be obtained, but background cells become complex and detection accuracy is difficult to ensure
Solution Approach 1:
The patent extracts and isolates circulating tumor cells (CTCs) from the complex blood background using automated capture technology. By specifically targeting CTCs through antibody-based capture and flow cytometry sorting, the system removes interfering background cells while preserving the target population, thereby improving detection accuracy.
Solution Approach 2:
The patent uses fluorescently labeled antibodies as intermediaries to specifically bind and identify CTCs. These antibodies serve as mediators that distinguish target cells from background cells based on surface marker expression, enabling accurate detection despite the complexity of the blood sample matrix.
3Quantity of substance
If membrane filtering is used to obtain circulating tumor cells from peripheral blood, then CTCs can be separated, but additional HE staining and expert reading are required which are cumbersome and subjective
Solution Approach 1:
The patent combines cell separation, staining, and detection into a single automated flow cytometry workflow. The system integrates multiple functions (filtration, immunostaining, fluorescence detection, and data analysis) into one continuous automated process, eliminating the need for separate HE staining and expert interpretation steps.
Solution Approach 2:
The patent uses fluorescent labeling with distinct emission wavelengths to automatically identify and characterize CTCs. Different fluorophores mark different cell surface markers, allowing the automated system to distinguish CTCs from background cells based on their fluorescence signature, replacing subjective visual assessment with objective optical detection.
4Measurement precision
If expert reading is used to identify CTCs with PD-L1 expression, then accurate identification can be achieved, but strong subjectivity and professionalism requirements make it difficult to promote and use
Solution Approach 1:
The patent replaces expert visual interpretation with automated flow cytometry analysis. The system uses computer-based algorithms to analyze fluorescence data, identify positive cells, and calculate PD-L1 expression levels automatically. This substitution maintains high identification accuracy while eliminating the need for expert pathology interpretation.
Solution Approach 2:
The automated system performs self-analysis of the stained cells, automatically gating populations, identifying positive cells based on fluorescence intensity thresholds, and generating quantitative results. This self-service capability eliminates dependency on expert operators, making the method accessible to laboratories without specialized pathology expertise.
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 significantly improves detection accuracy and reliability by isolating tumor cells from background cells, reducing false positives, and simplifying the detection process, thereby enabling more precise identification of PD-L1 expression on CTCs.
Implementation Method 1
a capture screen with EpCAM antibodies to specifically capture tumor cells from body fluids
Implementation Method 2
multicolor imaging analysis using DAPI, CK, and CD45 fluorescent antibodies
Data Source
Figure 1a~3b
Figure 4a~5b
AI summary
Disclosed is a method for detecting a tumor cell surface marker molecule, PD-L1, which method comprises the following steps: A, providing a capture screen that comprises a mesh matrix body and an EpCAM antibody formed in the mesh matrix body by means of incubation; B, making nucleated cells separated from the body fluid flow through the capture screen, such that tumor cells in the nucleated cells bind to the capture screen; C, fixing the captured tumor cells on the capture screen by using formaldehyde; D, successively using a PD-L1 primary antibody solution, a PD-L1 secondary antibody solution labeled with a fluorophore AlexaFluor 647, a pan-CK-AlexaFluor 488 primary antibody solution, a CD45 primary antibody solution and a CD45 secondary antibody solution labeled with a fluorophore AlexaFluor 568, to incubate the cells fixed on the capture screen, and then labeling all the cells on the capture screen with a nuclear fluorescent dye. Compared with existing methods, the detection method of the present invention can improve detection accuracy and is easier in terms of operation.