# US 10,502,678 B2 · Systems and methods for panel design in flow cytometry > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,502,678 B2 - Document ID: US10502678B2 - Status: Granted - Kind: B2 - Application number: 14/776,891 - Filed: 2014-03-14 - Published: 2016-01-28 - Granted: 2019-12-10 - Earliest claimed: 2013-03-15 - Assignees: Beckman Coulter, Inc. - Applicants: Beckman Coulter, Inc. - Inventors: Michael Kapinsky - Web page: https://patentgrep.com/patent/US10502678B2 ## CPC - G01N 21/255: {Details, e.g. use of specially adapted sources, lighting or optical systems} - G01N 15/14: Optical investigation techniques, e.g. flow cytometry - G01N 15/1425: {using an analyser being characterised by its control arrangement} - G01N 15/1434: Optical arrangements - G01N 15/01: specially adapted for biological cells, e.g. blood cells (investigating sedimentation of particle suspensions in blood G01N15/05) - G01N 2015/1006: {for cytology} - G01N 2015/1402: {Data analysis by thresholding or gating operations performed on the acquired signals or stored data} - G01N 2015/1477: {Multiparameters} - G01N 2201/12: Circuits of general importance; Signal processing - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Embodiments of the present invention encompass systems and methods for determining detection limits for various antibody-dye conjugates for flow cytometry. Exemplary techniques involve a linear superpositioning approach of spillover-induced enlargements of normally distributed measurement errors. ## Claims ### Claim 1 A method of designing a probe panel for differentiating populations of particles in a sample using a flow cytometer, the method comprising: determining a distortion factor that quantifies spillover effect caused by emission of a first label, intended to be measured in a first channel, into a second channel; determining a maximum expected signal of a first probe-label combination including the first label and a first probe, wherein the first probe binds to a first antigen associated with a population of particles of interest in the sample, and wherein the maximum expected signal is determined based on a brightness of the first label and a density of the first antigen on or inside the particles of interest in the sample; calculating in a detection limit of a second antigen detected in the second channel, wherein calculating the detection limit comprises multiplying the distortion factor by the maximum expected signal of the first probe-label combination, and wherein the detection limit is a threshold of signal intensity dividing particles considered to be positive for the second antigen from particles considered to be negative for the second antigen; selecting a probe-label combination to include in the probe panel based on the calculated detection limit; and differentiating the populations of particles in the sample using the probe panel in the flow cytometer. ### Claim 2 (depends on 1) The method of claim 1, wherein the distortion factor is an estimate of an increase in detection limit of the second antigen in the second channel as a function of an emission intensity of the first probe-label combination. ### Claim 3 (depends on 2) The method of claim 2, wherein the increase in detection limit of the second antigen in the second channel is caused by an increase in a measurement error as a function of the emission intensity of the first probe-label combination. ### Claim 4 (depends on 2) The method of claim 2, wherein the distortion factor is calculated using a crosstalk index. ### Claim 5 (depends on 1) The method of claim 1 wherein the distortion factor is mathematically modified by a coefficient representing a coexpression pattern of the first and second antigens. ### Claim 6 (depends on 1) The method of claim 1 further comprising: determining a distortion factor for each label in a first potential probe panel to calculate a total increase in detection limit in the second channel. ### Claim 7 (depends on 6) The method of claim 6, wherein selecting the probe-label combination is based on a comparison of the calculated total increase in detection limit with an expected minimum signal of a second probe-label combination, corresponding to the second antigen, in the second channel. ### Claim 8 (depends on 6) The method of claim 6, further comprising calculating a total increase in detection limit for each probe in the first potential probe panel. ### Claim 9 (depends on 8) The method of claim 8, further comprising: calculating a total increase in detection limit for each probe in a second potential probe panel; and selecting the probe panel based on a comparison of the calculated total increase in detection limit for each probe in the first potential probe panel with the calculated total increase in detection limit for each probe in the second potential probe panel. ### Claim 10 (depends on 8) The method of claim 8, further comprising: calculating a total increase in detection limit for each probe in a second potential probe panel; and selecting the probe panel based on the calculated total increase in detection limit for a prioritized probe in the first potential probe panel and the second potential probe panel. ### Claim 11 A method of designing a probe panel for differentiating populations of particles in a sample using a flow cytometer, the method comprising: determining a distortion factor that quantifies spillover effect caused by emission of a first label, intended to be measured in a first channel, into a second channel; determining a maximum expected signal of a first probe-label combination including the first label and a first probe, wherein the first probe binds to a first antigen associated with a population of particles of interest in the sample, and wherein the maximum expected signal is a function of a brightness of the first label and an expression pattern of the first antigen; calculating a detection limit of a second antigen detected in the second channel, wherein calculating the detection limit comprises multiplying the distortion factor by the maximum expected signal of the first probe-label combination, and wherein the detection limit is a threshold of signal intensity dividing particles considered to be positive for the second antigen from particles considered to be negative for the second antigen; selecting a probe-label combination to include in the probe panel based on the calculated detection limit; and differentiating the populations of particles in the sample using the probe panel in the flow cytometer. ### Claim 12 (depends on 11) The method of claim 11, wherein the expression pattern of the first antigen includes a mean density of the first antigen on or inside the particles of interest in the sample. ### Claim 13 (depends on 11) The method of claim 11, wherein the expression pattern of the first antigen includes a range of expression of the first antigen in the particles of interest in the sample. ### Claim 14 (depends on 11) The method of claim 11, wherein the distortion factor is mathematically modified by a coefficient representing a coexpression pattern of the first and second antigens. ### Claim 15 (depends on 14) The method of claim 14, wherein the coefficient is either one or zero. ### Claim 16 (depends on 15) The method of claim 15, wherein the coefficient is zero if the coexpression pattern of the first and second antigens is mutually exclusive. ### Claim 17 (depends on 15) The method of claim 15, wherein the coefficient is zero if the first antigen is a descendent of the second antigen. ### Claim 18 A method of designing a probe panel for differentiating populations of particles in a sample using a flow cytometer, the method comprising: determining a distortion factor that quantifies spillover effect caused by emission of a first label, intended to be measured in a first channel, into a second channel; determining a maximum expected signal of a first probe-label combination including the first label and a first probe, wherein the first probe binds to a first antigen associated with a population of particles of interest in the sample, and wherein the maximum expected signal is a function of the brightness of the first label and an expression pattern of the first antigen; calculating a detection limit of a second antigen detected in the second channel using a second probe-label combination including a second label and a second probe corresponding to the second antigen, wherein calculating the detection limit comprises multiplying the distortion factor by the maximum expected signal of the first probe-label combination, and wherein the detection limit is represented as a slope of a graph of emission measured in the second channel as a function of emission measured in the first channel; selecting a probe-label combination to include in the probe panel based on the calculated detection limit; and differentiating the populations of particles in the sample using the probe panel in the flow cytometer. ### Claim 19 (depends on 18) The method of claim 18, wherein calculating the detection limit is based on a linear superpositioning model of CV enlargements. ### Claim 20 (depends on 18) The method of claim 18, wherein the distortion factor is a measure of CV enlargements caused by color compensation.