Rare Circulating Cell Analysis via Fluorescence Quenching
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Solution Overview
Problem
Current methods for diagnosing diseases like cancer and cardiovascular diseases using rare circulating cells (RCCs) face challenges due to the lack of sensitive and accurate assays for identifying, enumerating, and analyzing these cells, particularly due to their low abundance and heterogeneity, which limits their utility as biomarkers.
Innovation Solution
A method for analyzing rare circulating cells in non-enriched blood samples involves detecting RCCs using immunofluorescent markers, assessing morphology, and quenching the immunofluorescence of detection markers to enable subsequent analysis of RCCs with high sensitivity and accuracy, allowing for detailed cellular and molecular analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If immunocapture technologies are used to enrich RCCs, then sensitivity of detection is improved, but the ability to perform unbiased multi-parametric analysis is lost
Solution Approach 1:
The patent removes the immunomagnetic bead-based capture step from the workflow, extracting the enrichment function to allow direct analysis of RCCs in non-enriched samples. This enables multi-parametric analysis without the bias introduced by targeted enrichment, while maintaining detection sensitivity through direct imaging of rare cells against abundant background cells.
Solution Approach 2:
The patent creates a universal platform that can perform multiple functions: detection, enumeration, and multi-parametric analysis of heterogeneous RCC populations simultaneously. The system uses morphology-based identification combined with multiple fluorescent markers to achieve both sensitivity and versatility in a single workflow.
2Ease of operation
If flow cytometry is used for cell sorting, then cell separation capability is improved, but the ability to robustly enumerate very small cell populations is lost
Solution Approach 1:
The patent replaces the mechanical flow cytometry sorting system with an imaging-based detection system. This substitution allows for robust enumeration of very small cell populations (1-10 cells/ml) by directly visualizing and counting rare cells in their native context, while still enabling subsequent sorting of identified cells for further analysis.
3Measurement precision
If immunofluorescent markers are used for detection, then detection sensitivity is improved, but the ability to perform downstream molecular analysis is compromised
Solution Approach 1:
The patent applies a quenching step that removes immunofluorescent signal from detection markers after imaging, allowing the same cells to then undergo downstream molecular analysis. This discarding of the fluorescent signal (without discarding the cells) enables both sensitive detection and subsequent molecular characterization of the same RCC population.
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 robust and sensitive detection and analysis of RCCs, providing a deeper understanding of their biology, which can improve disease diagnosis and personalized treatment plans by retaining a significant number of cells for further analysis while maintaining their morphological characteristics.
Implementation Method 1
quenching the immunofluorescence of the one or more immunofluorescent RCC detection markers including contacting the RCCs with a quenching buffer, wherein the immunofluorescence is quenched by more than 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 99.99%
Data Source
AI summary
The disclosure provides methods for analyzing rare circulating cells (RCCs) at cellular and molecular level following their detection in non-enriched blood samples, methods of this disclosure serve as diagnostic methods for several disease conditions, including cardiovascular diseases and cancer.