Flow Cytometry RNA Assay for Rapid T-Cell Activation Detection

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Solution Overview

Problem

Current immunodiagnostic assays fail to rapidly distinguish between stable latent and early active Mycobacterium tuberculosis infection, unable to detect rare or transient cell states associated with disease stage and evolution, and lack the sensitivity to identify low-frequency T cell populations in small samples of peripheral blood.

Innovation Solution

A method for detecting RNA molecules in individual cells by providing a sample, inducing gene expression with specific compounds, labeling RNA molecules with fluorescent probes, and using flow cytometry for detection, allowing for the identification of cytokine gene expression such as IL-2, TNFα, or IFNγ.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If current immunodiagnostic assays are used, then they can recognize the presence of pathology, but they fail to rapidly distinguish between stable latent and early active tuberculosis infection

Engineering Contradiction:

Improveability to distinguish infection stagesVSAvoiddiagnostic time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent extracts and measures specific RNA molecules (such as cytokine mRNAs) from individual T cells to detect early active tuberculosis infection. By focusing on specific molecular markers rather than general immune responses, the assay rapidly distinguishes between latent and active infection stages with high precision.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent replaces traditional mechanical/enzymatic detection methods (such as ELISA or Western blot) with flow cytometry-based fluorescent detection. This substitution enables rapid, sensitive measurement of RNA molecules in individual cells, providing stage-specific diagnostic information within hours rather than days.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If ensemble measurements are used, then they provide average responses, but they escape detection of rare or transient cell states associated with disease stage

Engineering Contradiction:

Improvedetection of rare cell statesVSAvoidmeasurement complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent segments the immune response measurement into individual T cell analyses rather than measuring bulk population averages. Flow cytometry enables examination of each cell's RNA expression profile separately, revealing rare or transient cell states that would be masked in ensemble measurements.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent uses fluorescently labeled hybridization probes that produce color changes (fluorescence signals) to detect specific RNA molecules in individual T cells. Different fluorophores allow simultaneous detection of multiple RNA targets, enabling complex immune response characterization through color-coded flow cytometry readouts.

Inventive Principle:

Principle #32Color changes

3Reliability

If standard microbiological diagnosis is used, then it detects individuals with tubercle bacilli in respiratory secretion, but it fails to detect early active disease before transmission

Engineering Contradiction:

Improvediagnostic reliabilityVSAvoidtime to diagnosis

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent performs preliminary detection of T cell RNA responses to mycobacterial antigens before clinical symptoms or bacillary shedding occur. By measuring early immune activation markers (such as cytokine mRNAs) in peripheral blood T cells, the assay identifies early active disease at a stage when transmission risk is low but intervention is still effective.

Inventive Principle:

Principle #10Preliminary action

4Adaptability or versatility

If current assays are used, then they provide general pathology detection, but they fail to assess stage-specific expression of mycobacterial antigens

Engineering Contradiction:

Improvestage-specific detection capabilityVSAvoidsample requirements

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The patent creates a universal flow cytometry-based platform that can detect multiple RNA targets (different cytokines, activation markers) in a single assay. This multi-functional system adapts to detect various mycobacterial antigen responses and immune activation states using the same basic methodology and minimal sample requirements.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 rapid and sensitive measurement of gene expression changes in individual cells, characterizing immune responses and disease progression, and can detect low-frequency T cell populations, improving diagnostic capabilities for tuberculosis and other immunopathological conditions.

Implementation Method 1

labeling copies of at least one RNA molecule expressed by the cells with a set of fluorescently labeled oligonucleotide hybridization probes

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS11767551B2Rapid assays for T-cell activation by RNA measurements using flow cytometry

Publication Date: 2023.09.26 RUTGERS THE STATE UNIV

AI summary

The present invention relates to a method for rapidly detecting copies of at least one RNA molecule expressed in individual cells and uses thereof.