Full Spectrum Flow Cytometer 13-Color B Cell Panel
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Solution Overview
Problem
Conventional flow cytometry is limited in its ability to perform high-throughput, in-depth analysis of immune cell populations due to the need for multiple tubes and redundant markers, which complicates the characterization of immune subpopulations and is inefficient with limited sample availability.
Innovation Solution
The development of a full spectrum flow cytometer that measures fluorochrome emission across multiple lasers using many detectors, allowing for the combination of 30 or more fluorescently labeled antibodies and enabling the creation of highly multiparametric panels, such as 28 and 40-color panels, to achieve detailed characterization of immune cells from a single sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional flow cytometry uses multiple tubes and redundant markers to analyze immune cell populations, then measurement precision can be maintained, but device complexity and sample consumption increase significantly
Solution Approach 1:
The patent combines multiple flow cytometry analyses into a single tube by using a panel of fluorescently labeled antibodies with spectrally resolved detection. Instead of running separate tubes for different marker combinations, the invention integrates multiple antibody stains (including redundant markers) into one multiparameter panel that can be analyzed simultaneously, reducing sample consumption and operational complexity while maintaining measurement precision through spectral unmixing algorithms
Solution Approach 2:
The flow cytometry system is designed with multi-functional capability to detect multiple fluorochromes across different excitation wavelengths (355nm, 405nm, 488nm, 561nm, 640nm lasers) using a unified detection platform. The system can analyze various immune cell populations (B cells, T cells, NK cells, monocytes, granulocytes) and their subsets within a single experimental setup, eliminating the need for separate specialized assays for different cell types
2Loss of information
If conventional flow cytometry uses multiple tubes for different marker combinations, then analysis depth can be achieved, but productivity decreases due to increased sample processing time
Solution Approach 1:
The patent enables continuous analysis of multiple immune cell populations and subsets within a single sample tube by using a comprehensive antibody panel that simultaneously targets markers across different cell lineages. The spectral detection system continuously collects data from all fluorochrome-labeled antibodies in parallel, eliminating the need for sequential tube processing and enabling high-throughput characterization of B cells, T cells, and other immune subsets in a single uninterrupted run
Solution Approach 2:
The invention adds a spectral dimension to flow cytometry detection by resolving fluorochrome emissions across multiple wavelengths (355nm-640nm lasers with corresponding detectors). This spectral dimensionality allows the system to distinguish between multiple fluorochromes with overlapping emission spectra, enabling simultaneous detection of 20+ markers in a single tube without sacrificing analysis depth or requiring additional physical dimensions like multiple tubes
3Measurement precision
If conventional flow cytometry uses limited sample volume distributed across multiple tubes, then measurement precision is compromised, but device complexity remains manageable
Solution Approach 1:
The patent consolidates multiple antibody stains and cell population analyses into a single tube configuration, maximizing the utilization of limited sample volume. By combining B cell markers (CD19, CD20, CD27, IgD, IgM, IgG), T cell markers (CD3, CD4, CD8, PD-1, TIM-3), and other lineage-specific markers in one multiparameter panel, the system achieves comprehensive immune cell characterization while consuming minimal sample volume that would otherwise be distributed across multiple separate tubes
Solution Approach 2:
The invention changes the detection parameter space by implementing spectral resolution across multiple excitation wavelengths and corresponding emission detectors. This parameter expansion allows the system to distinguish between numerous fluorochrome-labeled antibodies within a single tube, maintaining measurement precision for rare cell populations without requiring increased sample volume, as the spectral dimension provides additional discriminatory power
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 the identification of up to 45 metaclusters from a single tube sample with outstanding resolution, providing a powerful tool for immune system characterization and biomarker discovery, even with limited sample availability.
Implementation Method 1
In modern flow cytometry, cells are fluorescently labelled and then excited by laser(s) to emit light at varying wavelengths.
Implementation Method 2
Those detector(s) in line with the light beam, and typically up to 20 degrees offset from the laser beam's axis, are used to measure Forward Scatter or FSC. Other detector(s) are placed perpendicular to the stream and are used to measure Side Scatter (SSC).
Implementation Method 3
The nozzle or cuvette can be cone shaped causing a narrowing of the sheath and subsequent increase in the fluid velocity. The sample is introduced into the center and is focused by the Bernoulli effect.
Implementation Method 4
Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes.
Data Source
AI summary
In one embodiment, a method of building an optimized color flow high parameter reagent 13-color panel for B cell monitoring for analyzing B cell subsets in anti-CD20 treated autoimmune patients using a full spectrum flow cytometer is disclosed. In another embodiment, a reagent kit for B cell monitoring for analyzing B cell subsets in anti-CD20 treated autoimmune patients is disclosed for use with a full spectrum flow cytometer.