Methods of forming multi-color fluorescence-based flow cytometry panel

Patent metadata

Field	Value
Publication number	US-12209948-B2
Application number	US-202117304843-A
Country	US
Kind code	B2
Filing date	Jun 26, 2021
Priority date	Jun 26, 2020
Publication date	Jan 28, 2025
Grant date	Jan 28, 2025

Abstract

In one embodiment, a method of building an optimized color flow cytometry panel is disclosed using a full spectrum flow cytometer with five excitation lasers and five corresponding detection modules. 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.

First claim

What is claimed is:

1. A method of building a color flow cytometry panel using a full spectrum laser flow cytometer, the method comprising:
selecting thirty (30) or more cell markers for biological cells of interest;
identifying fluorochromes to be used in the flow cytometry panel;
analyzing full spectrum of each fluorochrome across detectors in the full spectrum laser flow cytometer;
comparing spectra of combination of pairs of each of the commercially available fluorochromes by determining a similarity index for each pairing of fluorochromes;
selecting thirty (30) or more optimal fluorochromes using the similarity index and a complexity index for each of the fluorochromes;
calibrating the lasers and detectors in the flow cytometer;
pairing the thirty or more optimal fluorochromes with the thirty (30) or more selected cell markers, according to the brightness of the fluorochrome and the expression density of the cell marker;
staining the biological cells of interest with the antibody conjugated fluorochromes, comprising the thirty (30) or more optimal fluorochromes and antibody specific to the thirty (30) or more cell markers, to create a multicolor sample;
running the multicolor sample through the full spectrum flow cytometer;
receiving data from the detectors of the full spectrum flow cytometer; and
processing the received data using a computer processor to form the color flow cytometry panel.

2. The method of claim 1 , wherein the biological cells of interest are selected from a group consisting of CD4 T cells, CD8 T cells, regulatory T cells (Tregs), γδ T cells, NKT-like cells, B cells, NK cells, monocytes, and dendritic cells.

3. The method of claim 1 , wherein selecting the thirty (30) or more optimal fluorochromes comprises, selecting the fluorochromes based on peak emission wavelength spread across the five laser colors of the full spectrum flow cytometer.

4. The method of claim 1 , wherein selecting the thirty (30) or more optimal fluorochromes comprises, quantifying uniqueness of each of a group of sixty-five (65) fluorochromes.

5. The method of claim 4 , wherein selecting the thirty (30) or more optimal fluorochromes comprises, analyzing the spectra of each of the sixty-five (65) fluorochromes using the full spectrum flow cytometer.

6. The method of claim 5 , wherein selecting the thirty (30) optimal fluorochromes comprises,
comparing the spectra of each pairing of the sixty-five (65) fluorochromes; and
assigning a similarity index to each pairing of fluorochromes.

7. The method of claim 6 , wherein selecting the thirty (30) optimal fluorochromes further comprises,
determining a threshold similarity index value and not selecting at least one fluorochrome of the pair of fluorochromes with a similarity index value higher than the threshold similarity index value.

8. The method of claim 6 , wherein selecting the thirty (30) optimal fluorochromes comprises,
choosing the thirty (30) optimal fluorochromes with the lowest similarity index.

9. The method of claim 8 , wherein the lowest-similarity index value that will produce high resolution data is 0.98.

10. The method of claim 1 , wherein identifying the thirty (30) optimal fluorochromes comprises:
determining a complexity index of the group of thirty (30) fluorochromes;
determining a threshold complexity index above which the group of thirty (30) fluorochromes are not considered optimal.

11. The method of claim 10 , wherein the threshold complexity index is fifty-four (54).

12. The method of claim 1 , wherein pairing the thirty (30) or more optimal fluorochromes with the thirty (30) or more selected cell markers comprises;
assigning the dimmest fluorochromes to the highest expressing antigens;
assigning tertiary markers to bright fluorochromes; and
avoiding placing highly expressed antigens adjacent to co-expressed antigens with lower expression for fluorochromes with a same primary excitation laser or similar emission wavelengths.

13. The method of claim 1 , wherein processing the received data comprises:
manually gating to remove aggregates, dead cells, debris, and CD45 negative events;
dating traditionally defined peripheral blood mononuclear cell (PBMC) populations;
sub-sample the data to include only the CD45+ live singlets,
unmix data using software with an ordinary least squares algorithm
performing an optimal stochastic neighbor embedding (opt-SNE) analysis of the data; and
assembling clusters into commonly recognized biological populations and generate a heatmap of the resulting populations.

Assignees

• Cytek Biosciences Inc

Inventors

• Jaimes Maria
• Zhang Zhenyu
• Zhou Jian
• Gu Huimin
• Shao Qing
• Yan Ming
• Vrane David

Classifications

• G01N2015/018

  Platelets · CPC title

• G01N15/01

  specially adapted for biological cells, e.g. blood cells (investigating sedimentation of particle suspensions in blood G01N15/05) · CPC title

• G01N2015/1493

  Particle size · CPC title

• G01N2015/144

  Imaging characterised by its optical setup · CPC title

• G01N2015/1438

  Using two lasers in succession · CPC title