T Cell Activation Classification Using Autofluorescence Phasor Analysis
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Solution Overview
Problem
Current methods for determining T cell activation, such as flow cytometry and immunofluorescence imaging, require contrast agents and tissue fixation, making them unsuitable for use in treatments like CAR T cell therapy where labeled cells are needed.
Innovation Solution
A T cell classification device using a time-resolved autofluorescence decay spectrometer and phasor analysis to classify T cells based on their activation state without the need for contrast agents or fixation, employing a processor to compute phasor coordinates from autofluorescence decay signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometry or immunofluorescence imaging is used to determine T cell activation, then activation state can be detected, but contrast agents and tissue fixation are required which make the cells unsuitable for subsequent therapeutic use
Solution Approach 1:
The patent utilizes the cell's own autofluorescence properties (NADH and FAD) as natural contrast agents to detect T cell activation state. By measuring the intrinsic fluorescence of metabolic cofactors that change during activation, the system eliminates the need for external fluorescent labels and fixation procedures, thereby preserving cell viability for subsequent therapeutic applications
Solution Approach 2:
The patent detects T cell activation by monitoring changes in the fluorescence lifetime parameters of endogenous metabolites NADH and FAD. As T cells activate, their metabolic state changes, altering the fluorescence decay characteristics of these cofactors. This parameter-based detection method provides activation information without requiring physical or chemical modification of the cells
2Reliability
If time-resolved autofluorescence decay measurement is used, then cell viability is preserved for therapy, but the measurement system becomes more complex
Solution Approach 1:
The patent transitions from measuring steady-state fluorescence intensity to measuring fluorescence decay over time (adding the time dimension). By resolving the temporal profile of autofluorescence decay, the system can distinguish between different metabolic states of T cells based on the characteristic lifetime signatures of NADH and FAD, providing more information without requiring additional labels or complex sample preparation
3Measurement precision
If multiple frequencies are used for phasor analysis, then classification precision improves, but computational requirements increase
Solution Approach 1:
The patent calculates phasor coordinates at multiple frequency points (including the excitation frequency and its harmonics) to improve classification accuracy. By sampling the fluorescence decay signal at several discrete frequencies and converting to phasor space, the system creates a more robust signature for distinguishing activated from resting T cells, with the computational cost justified by the significant improvement in classification precision
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 accurate classification of T cells with high precision, allowing for the enrichment of activated T cells for treatments like CAR T cell therapy, while avoiding the use of fluorescent labels and fixation.
Implementation Method 1
acquire a time-resolved autofluorescence decay signal for a T cell positioned in the observation zone
Implementation Method 2
The time-resolved autofluorescence decay spectrometer is configured to acquire a time-resolved autofluorescence decay signal
Data Source
AI summary
Systems and methods for classifying T cells by activation state are disclosed. The system includes a cell analysis pathway, a time-resolved autofluorescence decay spectrometer, a processor, and a non-transitory computer-readable memory. The memory is accessible to the processor and has stored thereon instructions. The instructions, when executed by the processor, cause the processor to: a) receive the time-resolved autofluorescence decay signal; b) compute at least a first phasor coordinate at a first frequency and a second phasor coordinate at a second frequency from the time-resolved autofluorescence decay signal, wherein the first and second frequency are different; and c) compute an activation prediction for the T cell using at least the first phasor coordinate and the second phasor coordinate.