Becton Dickinson and Company Limited

Peter Mage

Keegan Owsley

David A Roberts

Lingjie Wei

Mirko Corselli

Shreyas Bhaban

Yina Wang

Peter Johnson

Howard Ray Seay

Aaron Ross

Rane Fields

Rick Hou

Wenyu Bai

Methods of identifying a fluorochrome panel suitable for use in a flow cytometric protocol are provided. Methods of interest include receiving an instrument identifier and a request for N fluorochrome identifiers, selecting two or more subsets of N fluorochrome identifiers from a set of fluorochrome identifiers in a spectral matrix associated with the instrument identifier, and identifying the fluorochrome panel from the two or more subsets of N fluorochrome identifiers based on a numerical stability metric calculated for each of the two or more subsets of N fluorochrome identifiers using the spectral signatures associated with the two or more subsets of N fluorochrome identifiers. Systems and non-transitory computer readable storage media for practicing the invention are also provided.

Methods and systems for identifying a fluorochrome panel

Aspects of the present disclosure include methods for spectrally resolving light from fluorophores having overlapping fluorescence spectra in a sample. Methods according to certain embodiments include detecting light with a light detection system from a sample having a plurality of fluorophores having overlapping fluorescence spectra and spectrally resolving light from each fluorophore in the sample. In some embodiments, methods include estimating the abundance of one or more of the fluorophores in the sample, such as on a particle. In certain instances, methods include identifying the particle in the sample based on the abundance of each fluorophore and sorting the particle. Methods according to some embodiments includes spectrally resolving the light from each fluorophore by calculating a spectral unmixing matrix for the fluorescence spectra of each fluorophore. Systems and integrated circuit devices (e.g., a field programmable gate array) for practicing the subject methods are also provided.

Methods for spectrally resolving fluorophores of a sample and systems for same

Methods of determining an ideal gain for a detector in a light detection system of a flow cytometer are provided. Methods of interest include irradiating a flow stream with light from a light source, incrementally increasing the gain of the detector such that the detector collects light from the flow stream at each of a plurality of successively increasing gains, obtaining a baseline noise level from the detector at each gain in the plurality of successively increasing gains, calculating a limit of detection (LoD) for each gain in the plurality of successively increasing gains, and assessing the calculated LoDs to determine the ideal gain. Systems and computer readable storage media for practicing the invention are also provided.

Methods and systems for determining an ideal detector gain

Aspects of the present disclosure include methods for determining baseline noise of a photodetector (e.g., in a light detection system of a particle analyzer). Methods according to certain embodiments include irradiating a sample having particles in a flow stream, detecting light with the photodetector from the irradiated flow stream, generating data signals from the detected light and calculating a moving average mean squared error of the generated data signals to determine the baseline of the photodetector. Systems (e.g., particle analyzers) having a light source and a light detection system that includes a photodetector for practicing the subject methods are also described. Integrated circuits and non-transitory computer readable storage medium are also provided.

Methods for continuous measurement of baseline noise in a flow cytometer and systems for same

Aspects of the present disclosure include methods for identifying a set of fluorophore-biomolecule reagent pairs for characterizing a sample by flow cytometry. Methods according to certain embodiments include calculating a spectral spillover spreading parameter for a plurality of fluorophores, pairing each fluorophore with a biomolecule that is specific for a biomarker of a cell in the sample to generate a plurality of fluorophore-biomolecule reagent pairs, generating an adjusted spillover spreading matrix for the fluorophore-biomolecule reagent pairs based on the spectral spillover spreading parameter of each fluorophore and a biomarker classification parameter and identifying an optimal set of fluorophore-biomolecule reagent pairs based on the calculated spillover spreading values from the adjusted spillover spreading matrix. Systems and non-transitory computer readable storage medium for practicing the subject methods are also provided.

Methods for flow cytometry panel design based on modeling and minimizing spillover spreading, and systems for practicing the same

Aspects of the present disclosure include methods for adjusting a particle classification index in response to one or more saturated data signals from light detected from particles in a flow stream. Methods according to certain embodiments include detecting light from particles in a flow stream; generating a plurality of data signals from the detected light; identifying one or more saturated data signals; generating a saturated signal index that corresponds to the identified saturated data signals; and applying the saturated signal index to a particle classification index to generate an adjusted particle classification index. In some embodiments, methods include determining one or more parameters of a particle (e.g., for use in a particle sort decision) by calculating an adjusted spectral unmixing matrix for the fluorescence of the particle that excludes one or more saturated data signals. Systems and integrated circuit devices (e.g., a field programmable gate array) for practicing the subject methods are also provided. Non-transitory computer readable storage mediums are also described.

Methods for identifying saturated data signals in cell sorting and systems for same

Aspects of the present disclosure include methods for adjusting sensitivity of a photodiode in a light detection system. Methods according to certain embodiments include determining a background data signal from a photodetector at a plurality of detector gains, irradiating the photodetector with a light source at a plurality of different light intensities, generating data signals from the photodetector for the plurality of light intensities at each detector gain and adjusting the photodetector to the detector gain that corresponds to the lowest light irradiation intensity that generates a data signal resolvable from the background data signal. Systems (e.g., particle analyzers) having a light source and a light detection system that includes a photodetector for practicing the subject methods are also described. Non-transitory computer readable storage medium are also provided.

Methods for determining detector gain in a flow cytometer

Aspects of the present disclosure include methods for flow cytometrically sorting a sample with particles, such as cells, based on order of identification. Methods according to certain embodiments include introducing the sample into a flow cytometer; flowing the introduced sample in a flow stream; irradiating the sample in the flow stream with a light source; detecting light from cells in the sample flowing in the flow stream; identifying phenotypes of cells in the sample flowing in the flow stream based on one or more data signals generated from the detected light; and dynamically sorting into partitions cells of the sample that have a phenotype of a collection of predetermined phenotypes based on order of identification. Systems for practicing the subject methods are also provided. Non-transitory computer readable storage mediums are also described.

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San Jose, CA, United States of America

Peter Mage