A method and system for evaluating the collinearity of multiple autofluorescence spectra of a sample.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- BECTON DICKINSON & CO
- Filing Date
- 2025-10-24
- Publication Date
- 2026-06-23
Smart Images
Figure 2026102440000001_ABST
Abstract
Claims
1. In a flow cytometer, irradiating a sample containing particles in the flow stream with a light source, A photodetection system equipped with a photodetector detects light from the irradiated particles, To measure the autofluorescence spectrum generated by the particles in the sample, To evaluate the collinearity between the autofluorescence spectra generated by two or more different particles in the sample. Methods that include...
2. The method according to claim 1, comprising selecting a group of autofluorescence spectra for evaluating collinearity.
3. Selecting the aforementioned group of autofluorescence spectra is a) To generate a scatter plot including the fluorescence parameters of the particles of the sample, and Gating one or more groups on the scatter plot based on the median fluorescence intensity measured for each of the aforementioned particles, or b) Identifying the particle population by applying an unsupervised clustering algorithm, or c) Applying statistical analysis algorithms The method according to claim 2, including the method described in claim 2.
4. The method includes evaluating the unmixing performance of two or more autofluorescence spectra generated by the particles in the sample, and the evaluation of the unmixing performance is A step of generating spectral matrices relating to the fluorescence spectra of one or more fluorescent dyes and one or more autofluorescence spectra generated by the particles in the sample, Applying the aforementioned spectral matrix to unmix the fluorescence spectra generated by the unstained control, single-stained control, stained sample, or any combination thereof, Calculate one or more of the unmixing bias and unmixing variance. The method according to any one of claims 1 to 3, including
5. The method according to claim 4, wherein calculating the unmixing bias includes measuring the presence of a false-positive unmixed fluorescent dye signal associated with the autofluorescence spectrum.
6. Evaluating the aforementioned collinearity To generate a spectral matrix related to the autofluorescence generated by the particles in the sample, The inverse matrix is calculated from the generated spectral matrix, To identify the autofluorescence spectrum related to the dispersion of the data generated by the flow cytometer using the autofluorescence spectrum, and The method according to any one of claims 1 to 5, including the method described in any one of claims 1 to 5.
7. The method according to any one of claims 1 to 6, further comprising identifying the autofluorescence spectrum generated by the particles in the sample, which minimizes unmixing bias and minimizes the variance of the generated data.
8. The method according to any one of claims 1 to 7, further comprising generating a visualization of the evaluated collinearity of the autofluorescence spectrum in the generated data.
9. The method according to claim 8, wherein the visualization includes a panel hotspot matrix.
10. The method according to any one of claims 1 to 9, wherein evaluating the collinearity between the autofluorescence spectra includes evaluating the dispersion resolution (VDP).
11. The method according to any one of claims 1 to 10, further comprising removing the autofluorescence spectrum which has been determined to be collinear.
12. The method according to any one of claims 1 to 11, further comprising removing the autofluorescence spectrum that contributes to the maximum amount of variance in the flow cytometer data, and removing the autofluorescence spectrum that contributes to the maximum amount of unmixing bias in the spectral unmixing of the flow cytometer data.
13. A light source for irradiating a sample containing particles in a flowstream, A photodetection system comprising a photodetector for detecting light from the irradiated particles, A processor having memory, wherein the memory is operablely coupled to the processor and includes stored instructions, and when an instruction is executed by the processor, the processor has The autofluorescence spectrum generated by the particles in the sample is measured. To evaluate the collinearity between the autofluorescence spectra generated by two or more different particles in the sample. Processor and A flow cytometer system equipped with the following features.
14. The aforementioned memory, collinear, or Does it contribute to the maximum amount of variance in flow cytometer data, or This contributes to the maximum amount of unmixing bias in spectral unmixing of flow cytometer data. The flow cytometer system according to claim 13, comprising an instruction to remove the autofluorescence spectrum determined to be the same.
15. A non-temporary computer-readable storage medium containing stored instructions, wherein the instructions are An algorithm for irradiating a sample containing particles in a flow stream with a light source in a flow cytometer, An algorithm for detecting light from irradiated particles in a photodetector equipped with a photodetector, An algorithm for measuring the autofluorescence spectrum generated by the particles in the sample, An algorithm for evaluating the collinearity between autofluorescence spectra generated by two or more different particles in the sample, Non-temporary computer-readable storage media, including [specific type of storage medium].