Microbead Kit and Method for Quantitative Calibration and Performance Monitoring of a Fluorescence Instrument

Abstract

Fluorescently labeled microsphere calibration and quality control particles for use in establishing standardized fluorescence detector setup, monitoring daily QC of flow cytometers and quantitative calibration of the fluorescence detectors in traceable units of measure (molecules equivalent soluble fluorescence; MESF).

What is claimed is: 
     
         1 . A calibration kit comprising:
 a. a set of populations of microbeads;   b. each of said microbead populations having a median fluorescence intensity within a linear detection range of a fluorescence detector to be calibrated;   c. each of said microbead populations being spectrally different and determinable from other microbead populations in the same set to allow discrimination thereof;   d. at least two microbead populations of said set having spectrally different and determinable quantities of one or more internally-bound fluorophores measurable by a fluorescence detector to be calibrated; and   e. at least one microbead population of said set, comprising a spectrally different and determinable quantity of one or more fluorophores bound to a surface of the microbeads in the microbead population, measurable by a fluorescence detector to be calibrated.   
     
     
         2 . A microbead calibration kit according to  claim 1 , wherein the microbead populations are provided in a single liquid suspension. 
     
     
         3 . A microbead calibration kit according to  claim 1 , wherein at least one microbead population contains a determinable quantity of one or more spectrally different and determinable fluorophores measurable by a fluorescence detector to be calibrated, and has spectral properties that are sufficiently different from an experimental sample to allow reliable discrimination thereof, and is provided in a separate suspension to allow mixing with the experimental sample as an internal control. 
     
     
         4 . A microbead calibration kit according to  claim 1 , wherein said microbeads have a diameter in the range from about 0.5 microns to about 70 microns. 
     
     
         5 . A microbead calibration kit according to  claim 1 , wherein said microbeads have a refractive index in the range from about 1.2 to about 1.8. 
     
     
         6 . A microbead calibration kit according to  claim 1 , wherein said spectral properties are selected from the group consisting of: one or more different and determinable fluorophores, diameter, refractive index, reflectivity, fluorescence intensity, light scatter, and Raman spectrum. 
     
     
         7 . A microbead calibration kit according to  claim 1 , further comprising at least one unlabeled microbead population to serve as a control for autofluorescence of the microbead. 
     
     
         8 . A method for calibrating a flow cytometer or fluorescence microscope in terms of molecules equivalent soluble fluorescence (MESF), comprising the steps of:
 a. providing a microbead calibration kit as described in  claim 1 ;   b. measuring a detection reagent containing a fluorophore to be calibrated;   c. optionally comparing each microbead population to reference solutions of fluorophores to determine a mean equivalent number of reference fluorophores (ERF) or a mean MESF of each population;   d. comparing the detection reagent to reference solutions of the same fluorophore, to determine the mean MESF per molecule of detection reagent;   e. measuring the microbead populations; and   f. plotting a median fluorescence intensity (MFI) of the microbead populations against assigned calibration to obtain a slope and an intercept.   
     
     
         9 . The method of  claim 8  further comprising:
 using said mean MESF per molecule of detection reagent to calculate a modified regression equation written in terms of MFI versus number of molecules of detection reagent; and 
 using said modified regression equation to determine the number of molecules of said detection reagent bound to said experimental sample to form a value called molecules of detected analyte. 
 
     
     
         10 . The method of  claim 8 , wherein said detection reagent is selected from the group consisting of antibodies, peptides, nucleic acids, viability dyes, intercalating agents, lipid dyes, and carbohydrate polymers. 
     
     
         11 . The method of  claim 8 , wherein a computer program automatically identifies multiple microbead populations and calculates a regression equation for a calibrated fluorophore. 
     
     
         12 . The method of  claim 8 , further comprising mixing at least one microbead population in a well with the experimental sample. 
     
     
         13 . The method of  claim 9 , further comprising mixing one or more populations of microbeads with the experimental sample and measuring fluorescence intensity. 
     
     
         14 . The method of  claim 8 , wherein performance or data quality is monitored in real-time by monitoring fluorescence intensity of one or more microbead populations mixed with the experimental sample, and comparing said fluorescence intensity to an acceptable target range. 
     
     
         15 . The method for calibrating a flow cytometer or fluorescence microscope according to  claim 14 , wherein said at least two populations are compared by solution fluorimetry to reference solutions of fluorophore to determine the equivalent number of reference fluorophores of said populations. 
     
     
         16 . The method of  claim 8 , wherein said at least two populations are measured by flow cytometry to determine the intrinsic coefficient of variance of said populations, prior to the calibration of the flow cytometer or fluorescence microscope. 
     
     
         17 . The method of  claim 8 , wherein more than one population of microbeads containing surface-bound fluorophores are provided as separate liquid suspensions, and are mixed immediately prior to measurement. 
     
     
         18 . The method of  claim 8 , further comprising:
 adding at least one unlabeled microbead population to serve as a control for autofluorescence of the microbead;   measuring the at least one unlabeled microbead population; and   correcting for autofluorescence of the microbeads.   
     
     
         19 . A calibration kit comprising:
 a. a set of populations of microbeads, the microbeads having a surface;   b. each of said microbead populations having a median fluorescence intensity within a linear detection range of a fluorescence detector to be calibrated;   c. each of said microbead populations having spectral properties that are sufficiently different from any other microbead populations in the same set to allow discrimination thereof;   d. at least two microbead populations of said set comprising spectrally different and determinable quantities of one or more spectrally non-overlapping internally-bound fluorophores measurable by a fluorescence detector to be calibrated;   e. at least one microbead population of said set, having bound to the surface of each microbead, either directly or indirectly, a determinable quantity of one or more spectrally non-overlapping fluorophores measurable by a fluorescence detector to be calibrated; and   f. at least one unlabeled microbead population of said set, to serve as a control for autofluorescence of said material.   
     
     
         20 . A microbead calibration kit according to  claim 19 , wherein said microbeads have a diameter in the range from about 0.5 microns to about 70 microns. 
     
     
         21 . A microbead calibration kit according to  claim 19 , wherein said microbeads have a refractive index in the range from about 1.2 to about 1.8. 
     
     
         22 . A microbead calibration kit according to  claim 19 , wherein said spectral properties are selected from the group consisting of: one or more non-overlapping dyes, diameter, refractive index, reflectivity, fluorescence intensity, light scatter, and Raman spectrum. 
     
     
         23 . A method for calibrating a flow cytometer or fluorescence microscope in terms of molecules equivalent soluble fluorescence (MESF), comprising the steps of:
 a. providing a microbead calibration kit as described in  claim 1 ;   b. contacting an experimental sample with a detection reagent containing a fluorophore to be calibrated, said fluorophore being the same as the surface-bound fluorophores;   c. measuring the at least one unlabeled microbead population and using this value to correct for autofluorescence of the microbeads by subtracting autofluorescence;   d. comparing each microbead population by solution fluorimetry to reference solutions of fluorophore to determine a mean number of equivalent reference fluorophores or a mean MESF of each population;   e. comparing the detection reagent by solution fluorimetry to reference solutions of the same fluorophore, to determine the mean MESF per molecule of detection reagent;   f. measuring the microbead populations;   g. plotting a median fluorescence intensity (MFI) of the microbead populations containing one or more non-overlapping internally-bound fluorophores versus a reference solution of a fluorophore to determine the MESF of each population, performing a linear regression analysis of said plot to form a linear regression line, and determining a slope of the regression line;   h. determining the MFI of a microbead population containing the surface-bound fluorophores, and identifying a point at which said MFI intercepts said regression line, calculating a y-intercept, and rewriting the regression equation in terms of MFI versus MESF for the fluorophores used in step (d);   i. using said mean MESF per molecule of detection reagent to calculate a modified regression equation written in terms of MFI obtained in step (h) versus number of molecules of detection reagent; and   j. using said modified regression equation to determine the number of molecules of said detection reagent bound to said experimental sample to form a value called molecules of detected analyte.   
     
     
         24 . The method of  claim 23 , wherein said detection reagent is selected from the group consisting of antibodies, peptides, nucleic acids, viability dyes, intercalating agents, lipid dyes, and carbohydrate polymers. 
     
     
         25 . The method of  claim 23 , wherein performance or data quality is monitored in real-time by monitoring fluorescence intensity of one or more microbead populations mixed with the experimental sample, and comparing said fluorescence intensity to an acceptable target range. 
     
     
         26 . The method for calibrating a flow cytometer or fluorescence microscope according to  claim 23 , wherein said at least two populations are compared by solution fluorimetry to reference solutions of fluorophore to determine the equivalent number of reference fluorophores of said populations. 
     
     
         27 . The method for calibrating a flow cytometer or fluorescence microscope according to  claim 23 , wherein said at least two populations are measured by flow cytometry to determine the intrinsic coefficient of variance of said populations, prior to the calibration of the flow cytometer or fluorescence microscope. 
     
     
         28 . The method for calibrating a flow cytometer or fluorescence microscope according to  claim 23 , wherein said at least one population is compared by solution fluorimetry to reference solutions of a fluorophore to determine a median MESF or the equivalent number of reference fluorophores of said population.