Method and system for evaluating fluorescent dye panels for use in the analysis of flow cytometry data.

Abstract

The present invention provides a method and system for evaluating the suitability of a fluorescent dye panel for use in generating flow cytometer data. [Solution] Negative and positive populations are obtained for each single-stain fluorescent dye in the fluorescent dye panel. A covariance matrix is ​​calculated for each of the negative and positive populations. An unmixing covariance matrix for the positive population is calculated for each fluorescent dye in the fluorescent dye panel, and a diffusion metric is calculated from the unmixing covariance matrix. Based on the calculated diffusion metric, it is identified that two or more fluorescent dyes are associated with the variance of the flow cytometer data. Two or more fluorescent dyes in the fluorescent dye panel are identified as being associated with the variance of the flow cytometer data generated using the fluorescent dye panel.

Claims

1. A method for identifying two or more fluorescent dyes in a fluorescent dye panel related to the dispersion of flow cytometer data, To obtain a negative population and a positive population for each of two or more single-staining fluorescent dyes in the aforementioned fluorescent dye panel, The covariance matrix is ​​calculated for each of the aforementioned negative group and the aforementioned positive group, The unmixing covariance matrix of the positive population is calculated for each fluorescent dye in the aforementioned fluorescent dye panel, The diffusion metric is calculated from the aforementioned unmixing covariance matrix, Based on the calculated diffusion metric, identify that the two or more fluorescent dyes are associated with the dispersion of flow cytometer data. Methods that include...

2. The method according to claim 1, further comprising calculating a spillover diffusion matrix corresponding to the fluorescent dye panel.

3. The method according to any one of claims 1 to 2, comprising obtaining a negative population and a single positive population for each of the single staining fluorescent dyes in the fluorescent dye panel.

4. The method according to claim 3, wherein the negative group and the positive group are obtained from a reference database.

5. The method according to any one of claims 1 to 4, wherein the negative population includes a reference unstained sample.

6. The method according to any one of claims 1 to 5, wherein calculating the covariance matrix further comprises calculating the median signal vector for each of the negative and positive populations.

7. The unmixing covariance matrix of the positive population is calculated for each fluorescent dye in the fluorescent dye panel according to the following calculation: V Ｆ =M ｐｉｎｖ ×V Ｙ ×M ｐｉｎｖ Ｔ Here, V Ｆ However, this is the covariance matrix of the unmixed fluorescent dye signals, V Ｙ However, this is the covariance matrix of the raw photodetector measurements, M ｐｉｎｖ The method according to any one of claims 1 to 6, wherein the spectral matrix used for unmixing is the Moore-Penrose pseudoinverse matrix.

8. The method according to any one of claims 1 to 7, further comprising optimizing the fluorescent dye panel based on an evaluation of the suitability of the fluorescent dye panel for use in generating the flow cytometer data.

9. The method according to any one of claims 1 to 8, wherein the flow cytometer data includes a number of dimensions equal to the number of fluorescent dyes in the plurality of fluorescent dyes.

10. The method according to any one of claims 1 to 9, wherein the flow cytometer data is spectrally unmixed flow cytometer data or compensated flow cytometer data.

11. The method according to any one of claims 1 to 10, wherein the variance includes noise in the flow cytometer data.

12. The method according to any one of claims 1 to 11, further comprising generating a visualization of the evaluated suitability of the fluorescent dye panel for use in generating the flow cytometer data.

13. A method for identifying two or more fluorescent dyes in a panel of fluorescent dyes related to the dispersion of flow cytometer data, comprising determining the collinearity between the spectra of each of the two or more fluorescent dyes, at least in part, based on the dispersion resolution ratio (VDP) of spectra exceeding a set threshold.

14. A system for identifying two or more fluorescent dyes in a fluorescent dye panel related to the dispersion of flow cytometer data, comprising a processor having memory, the memory being operably coupled to the processor and containing stored instructions, and when the instructions are executed by the processor, the processor For one or more single-staining fluorescent dyes in the aforementioned fluorescent dye panel, a negative group and a positive group are obtained. The covariance matrix is ​​calculated for each of the aforementioned negative group and the aforementioned positive group. For each fluorescent dye in the aforementioned fluorescent dye panel, calculate the unmixing covariance matrix of the positive population. The diffusion metric is calculated from the aforementioned unmixing covariance matrix. A system that, based on the calculated diffusion metric, identifies that the two or more fluorescent dyes are associated with the dispersion of flow cytometer data.

15. A system for identifying two or more fluorescent dyes in a fluorescent dye panel related to the dispersion of flow cytometer data, comprising a processor having memory, the memory being operably coupled to the processor and comprising stored instructions, the system causing the processor to determine the collinearity between the spectra of each of the two or more fluorescent dyes, at least in part, based on a spectral dispersion resolution ratio (VDP) above a set threshold, when the instructions are executed by the processor.

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