US-20260126370-A1 - FLUORESCENCE SENSITIVITY MONITOR FOR A FLOW CYTOMETER

US20260126370A1US 20260126370 A1US20260126370 A1US 20260126370A1US-20260126370-A1

Abstract

Systems and methods for fluorescence sensitivity monitoring in a flow cytometer include measuring fluorescence of a sheath fluid to determine a sheath noise and setting a threshold detection value using the sheath noise. Fluorescence of a plurality of samples having predetermined fluorescence intensities at different wavelengths is measured, using the threshold detection value. Each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths is gated and, using the gating, a mean fluorescence intensity (MFI) is identified for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths. One or more fluorescence channels of the flow cytometer are calibrated by calculating, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, molecules of equivalent soluble fluorochrome (MESF) using the MFI and the sheath noise.

Inventors

Evgenia M. KIM
Maria C. GENTILE

Assignees

BECKMAN COULTER, INC.

Dates

Publication Date: 20260507
Application Date: 20250131

Claims (15)

1 . A method of fluorescence sensitivity monitoring in a flow cytometer, the method comprising: measuring fluorescence of a sheath fluid to determine a sheath noise; setting a threshold detection value using the sheath noise; measuring fluorescence of a plurality of samples having predetermined fluorescence intensities at different wavelengths, using the threshold detection value; gating for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths; identifying, using the gating, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, a mean fluorescence intensity (MFI); and calibrating one or more fluorescence channels of the flow cytometer by calculating, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, molecules of equivalent soluble fluorochrome (MESF) using the MFI and the sheath noise.
2 . The method of claim 1 , further comprising filtering the sheath fluid.
3 . The method of claim 2 , wherein the sheath fluid is a 5 nm sheath fluid.
4 . The method of claim 1 , wherein the sheath noise is the MFI of the sheath fluid.
5 . The method of claim 1 , wherein the plurality of samples having predetermined fluorescence intensities at different wavelengths comprise a plurality of hard dyed beads.
6 . The method of claim 1 , wherein calculating MESF uses a calibration equation determined by: measuring fluorescence of a plurality of color reader beads with each color reader bead being associated with a known amount of a fluorophore; defining MFI for each plurality of color reader beads per each fluorescence channel; and defining the calibration equation for each fluorescence channel using a relationship between the MFI and the known amount of the fluorophore.
7 . The method of claim 1 , wherein identifying, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, the MFI, includes determining, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, a peak intensity associated with the sample.
8 . The method of claim 7 , further comprising determining a median and a standard deviation for each peak.
9 . A system for fluorescence sensitivity monitoring in a flow cytometer, the system comprising: a laser configured to emit light toward an interrogation location to produce fluorescent light signals from particles directed through the interrogation location in a fluid stream; a fluorescence detector configured to detect the fluorescent light signals from particles directed through the interrogation location; a controller in communication with the fluorescence detector and including at least one processor and a non-transitory memory storing instructions which, when executed by the controller, cause the controller to: measure, with the fluorescence detector, fluorescence of a sheath fluid to determine a sheath noise; set a threshold detection value using the sheath noise; measure, with the fluorescence detector, fluorescence of a plurality of samples having predetermined fluorescence intensities at different wavelengths, using the threshold detection value; gate for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths; identify, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, a mean fluorescence intensify (MFI); and calibrate one or more fluorescence channels of the flow cytometer by calculating, for each sample of the plurality of samples of predetermined fluorescent intensity or condition, molecules of equivalent soluble fluorochrome (MESF) using the MFI and the sheath noise.
10 . The system of claim 9 , further comprising a filter for filtering the sheath fluid.
11 . The method of claim 10 , wherein the sheath fluid is a 5 nm sheath fluid.
12 . The method of claim 9 , wherein the sheath noise is the MFI of the sheath fluid.
13 . The method of claim 9 , wherein the plurality of samples having predetermined fluorescence intensities at different wavelengths comprise a plurality of hard dyed beads.
14 . The method of claim 9 , wherein the instructions for calculating MESF uses a calibration equation determined by the controller executing further instructions stored on the non-transitory memory which cause the controller to: measure fluorescence of a plurality of color reader beads with each color reader bead being associated with a known amount of a fluorophore; define MFI for each plurality of color reader beads per each fluorescence channel; and define the calibration equation for each fluorescence channel using a relationship between the MFI and the known amount of the fluorophore.
15 . The method of claim 9 , wherein the instructions for identifying, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, the MFI, include further instructions stored on the non-transitory memory which cause the controller to determine, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths: a peak intensity associated with the sample; and a median and a standard deviation for each peak.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application is being filed on Jan. 31, 2025, as a PCT International application and claims the benefit of and priority to U.S. Provisional Application No. 63/548,653, filed on Feb. 1, 2024, the disclosure of which is hereby incorporated by reference in its entirety. BACKGROUND Fluorescence sensitivity monitoring is used to ensure the accuracy and reliability of the fluorescence detectors. The primary purpose of monitoring fluorescence sensitivity is to guarantee the precise measurement of fluorescent signals emitted by cells or particles labeled with fluorochromes. This process involves regular quality control checks to maintain the instrument's performance and detect any deviations in sensitivity. By optimizing settings, such as the light detector voltages, for each fluorochrome, laboratories can enhance sensitivity without saturating signals, contributing to consistent and reliable experimental outcomes. SUMMARY Examples presented herein relate to a method of fluorescence sensitivity monitoring in a flow cytometer. The method includes measuring fluorescence of a sheath fluid to determine a sheath noise, setting a threshold detection value using the sheath noise, and measuring fluorescence of a plurality of samples having predetermined fluorescence intensities at different wavelengths, using the threshold detection value. The method further includes gating for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, identifying, using the gating, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, a mean fluorescence intensity (MFI), and calibrating one or more fluorescence channels of the flow cytometer by calculating, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, molecules of equivalent soluble fluorochrome (MESF) using the MFI and the sheath noise. In other examples presented herein, the method further includes filtering the sheath fluid. In further examples presented herein, the sheath fluid is a 5 nm sheath fluid. In other examples presented herein, the sheath noise is the MFI of the sheath fluid. In still other examples presented herein, the plurality of samples having predetermined fluorescence intensities at different wavelengths comprise a plurality of hard dyed beads. In yet other examples presented herein, calculating MESF uses a calibration equation determined by measuring fluorescence of a plurality of color reader beads with each color reader bead being associated with a known amount of a fluorophore, defining MFI for each plurality of color reader beads per each fluorescence channel, and defining the calibration equation for each fluorescence channel using a relationship between the MFI and the known amount of the fluorophore. In other examples presented herein, identifying, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, the MFI, includes determining, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, a peak intensity associated with the sample. In further examples presented herein, the method further includes determining a median and a standard deviation for each peak. Other examples presented herein relate to a method of determining a lower limit of detection on a flow cytometer. The method includes measuring fluorescence on a sheath fluid to determine a sheath noise, setting a threshold detection value using the sheath noise, and measuring fluorescence of a plurality of fluorescent samples using the threshold value. In other examples presented herein, the method further includes filtering the sheath fluid. In further examples presented herein, the sheath fluid is a 5 nm sheath fluid. In other examples presented herein, the sheath noise is the MFI of the sheath fluid. Still other examples presented herein relate to a method of calibrating one or more fluorescence channels of a flow cytometer. The method including measuring fluorescence of a plurality of samples having predetermined fluorescence intensities at different wavelengths, using a predetermined detection threshold value, gating for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, identifying, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, a mean fluorescence intensity (MFI), and calibrating one or more fluorescence channels of the flow cytometer by calculating, for each sample of the plurality of samples having predetermined fluorescence intensities at different wavelengths, molecules of equivalent soluble fluorochrome (MESF) using the MFI and the predetermined detection threshold. In other examples presented herein, the predetermined detect