Flow cytometry with data analysis for optimized dilution of fluid samples for flow cytometry investigation
Inventors
Gates, Tyler • Steaffens, Jeffrey
Assignees
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Publication Number
US-11137337-B2
Publication Date
2021-10-05
Expiration Date
Abstract
Flow cytometry investigation and flow cytometry result analysis to determine an optimized dilution factor range for flow cytometry investigation of a target sample fluid stock using a flow cytometer. The optimized dilution factor range may be determined using a screening assay module of a flow cytometry system to analyze flow cytometry results determined to fall within a dynamic range of a flow cytometer to determine the optimized dilution factor range for a given flow cytometer to investigate a given target sample fluid stock. In turn, a titer assay module may be executed to prepare particle titer results based on optimized target fluid samples provided within the optimized dilution factor range. The screening assay module and/or the titer assay module may provide automated data processing with data notifications and confirmations to provide robust data analysis.
Core Innovation
The document describes a flow cytometry method for quantification of target particles with particular particle attributes in a target sample fluid stock using a flow cytometer operable to flow fluid through an investigation zone subjected to investigation excitation radiation and to detect and measure response radiation. For a screening target set of screening target fluid samples comprising a dilution series of the screening sample fluid stock at a plurality of dilution factors, the method generates flow cytometry results including a quantification indication for the target particles. The screening target set results are stored in computer memory as data for later computational modules.
A screening assay module is executed by a processor to calculate corrected quantification indications by comparison of first selected flow cytometry results of the screening target set with corresponding second selected flow cytometry results of a screening control set, where the screening control set includes at least one screening blank control fluid sample corresponding to the screening target set. Based on analysis of the corrected quantification indications, the method determines an optimized sample dilution factor range for the screening stock fluid sample and saves the optimized sample dilution factor range in memory. An optimized target set of optimized target fluid samples is then formed by diluting the target sample fluid stock to dilution factors within the optimized sample dilution factor range.
The method executes a titer assay module by accessing the flow cytometry results for the optimized target set stored in memory and calculating particle titer results for the target sample fluid stock. The particle titer results are stored in the memory. The document further describes system components including a flow cytometer, a results database, memory-executable screening and titer modules, and a graphical user interface that can provide outputs and notifications related to dilution factors, fit, and titer results.
Claims Coverage
The independent claim set centers on 8 inventive features, with a two-stage screening-and-titer workflow that generates corrected quantification indications from a dilution series and derives an optimized sample dilution factor range before calculating particle titer results from an optimized target set.
Screening dilution series with corrected quantification indication comparison to blank control
Subjecting a screening target set of screening target fluid samples to first flow cytometry evaluation, wherein the screening target fluid samples comprise a dilution series of screening sample fluid stock diluted at a plurality of dilution factors; flowing each screening target fluid sample through the investigation zone under investigation excitation radiation; detecting response radiation to generate flow cytometry results including a quantification indication; storing the results in computer memory; and executing a screening assay module that calculates a corrected quantification indication by comparison of selected flow cytometry results of the screening target set with corresponding selected flow cytometry results of a screening control set including at least one screening blank control fluid sample, adjusting quantification indications based on the comparison.
Optimized sample dilution factor range determination from analysis of corrected quantification indications
Determining, based on analysis of the corrected quantification indications, an optimized sample dilution factor range for the screening stock fluid sample and saving the optimized sample dilution factor range in the memory.
Optimized target set flow cytometry and titer assay module particle titer calculation
Subjecting to second flow cytometry evaluation an optimized target set of optimized target fluid samples, each comprising the target sample fluid stock diluted to an optimized sample dilution factor within the optimized sample dilution factor range; flowing each optimized target fluid sample through the investigation zone under investigation excitation radiation; detecting response radiation to generate flow cytometry results including a quantification indication; storing the results in memory; and executing a titer assay module that accesses the flow cytometry results for the optimized target set, calculates particle titer results for the target sample fluid stock, and stores the particle titer results in the memory.
Linear regression fit on log-transformed corrected quantification indications for dilution quality
Determining a linear regression fit relative to log-transformed corrected quantification indications and multiple dilution factors, wherein one or more dilution quality parameters quantitatively characterize the linear regression fit.
Fit recalculation by excluding extreme dilution samples
Recalculating the fit by removing at least one screening target fluid sample at either the maximum said dilution factor or the minimum said dilution factor and determining the fit while excluding the corrected quantification indication for each removed sample.
Quantification correction notification based on target-versus-control indication comparison
Second-determining whether each quantification indication from the selected screening target set is greater than the corresponding quantification indication from the selected screening control set, and providing a quantification correction notification when the condition is not met.
Optimized performance concentration bounds define optimized sample dilution factor range
Defining the optimized sample dilution factor range using optimized performance concentration bounds such that a lower optimized concentration bound and an upper optimized concentration bound form an optimized performance sub-range.
Optimized versus screening sample selection with particle attribute constraints and fluorescent response detection
Defining an optimized target set and a screening target set across dilution factors with specified selection differences, with target particles of specified size, fluorescently stained screening/blank/optimized fluid samples, and detecting fluorescent emission response from labeled particles during flow through the investigation zone under investigation excitation radiation.
The claim set centers on generating corrected quantification indications by comparing screening dilution series results to blank control results, selecting an optimized sample dilution factor range from analysis of the corrected indications, and computing particle titer results from flow cytometry performed on the optimized target set. Dependent claims further specify fit-quality evaluation, notification logic based on target-versus-control comparisons, and additional constraints for optimized performance bounds and particle attribute/fluorescent response detection.
Stated Advantages
Not explicitly described in patent.
Documented Applications
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