Spectral Flow Cytometry 15-Color Leukocyte Panel
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Solution Overview
Problem
Current flow cytometry technologies face challenges in accurately enumerating leukocyte subsets in peripheral blood due to limitations in fluorochrome and marker selection, leading to incomplete characterization of immune subpopulations.
Innovation Solution
The development of a 15-color immunoprofiling panel using spectral flow cytometers, which enables precise identification of leukocyte subsets by analyzing the entire fluorochrome emission spectrum across multiple lasers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional flow cytometry with limited fluorochrome panels is used, then the number of markers that can be analyzed is restricted, but the system complexity and cost are lower
Solution Approach 1:
The patent transitions from conventional flow cytometry that measures intensity at a few discrete wavelengths to spectral flow cytometry that captures the entire emission spectrum at hundreds of wavelengths. This dimensional expansion from 2-4 parameters to hundreds of spectral parameters enables simultaneous analysis of 15+ fluorochromes and 30+ markers, resolving the contradiction between versatility and complexity by adding spectral dimensionality rather than simply adding more detectors.
Solution Approach 2:
The invention changes the measurement parameter from discrete intensity values at limited wavelengths to continuous spectral profiles across the entire emission range. By capturing full spectra and using spectral unmixing algorithms, the system can distinguish between fluorochromes with overlapping emission profiles, enabling high-plex analysis without proportionally increasing device complexity.
2Measurement precision
If spectral flow cytometry with 15-color panel is implemented, then characterization of immune subpopulations becomes more accurate, but the device complexity and data processing requirements increase
Solution Approach 1:
The patent introduces spectral unmixing algorithms as an intermediary computational layer that processes the complex spectral data. These algorithms deconvolve overlapping emission spectra by comparing against reference spectral profiles, enabling accurate identification of individual fluorochrome signals even when their emission spectra overlap significantly. This computational intermediary transforms the complex spectral measurements into precise marker quantification.
Solution Approach 2:
The invention replaces the mechanical/optical approach of using many separate detectors for each fluorochrome with a single spectrometer that captures all wavelengths simultaneously. The physical complexity of having 15+ separate detection channels is substituted with a unified spectral detection system combined with computational analysis, reducing optical complexity while maintaining or enhancing measurement precision.
3Ease of operation
If traditional flow cytometry panels are used, then the assay is simpler to perform, but the enumeration of leukocyte subsets becomes less accurate
Solution Approach 1:
The patent creates a universal spectral flow cytometry platform that can analyze multiple markers simultaneously using a single standardized instrument and data analysis pipeline. The spectral detection system and unmixing algorithms provide a multi-functional capability that handles 2-color to 15-color panels through the same technical approach, maintaining ease of operation while dramatically improving enumeration accuracy for leukocyte subsets.
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 allows for high-throughput, in-depth analysis of immune cells, providing more accurate and detailed characterization of immune subpopulations, which is essential for understanding immune responses and developing targeted therapeutic approaches.
Implementation Method 1
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
Implementation Method 2
A beam of laser light is directed at a hydrodynamically-focused stream of fluid that carries the cells
Implementation Method 3
Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors
Data Source
AI summary
In one embodiment, a method of building an optimized color flow cytometry panel is disclosed using a spectral flow cytometer with a least three excitation lasers and thirty-eight color detectors. In another embodiment, a graphical user interface is disclosed generated by a server computer from a fluorochrome database and displayed by a client computer to assist in the selection of a set of fluorochromes for use in an assay to analyze biological samples. The GUI can display spectra graphs to visually show how fluorochromes may overlap and can generate similarity indexes for the paired fluorochrome interference and a complexity index for overall many to many interferences generated by a selected group or set of fluorochromes.