WO2010090645A1 - Nouvelle génération de microbilles fluorescentes de référence comme substitut cellulaire - Google Patents

Nouvelle génération de microbilles fluorescentes de référence comme substitut cellulaire Download PDF

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Publication number
WO2010090645A1
WO2010090645A1 PCT/US2009/033519 US2009033519W WO2010090645A1 WO 2010090645 A1 WO2010090645 A1 WO 2010090645A1 US 2009033519 W US2009033519 W US 2009033519W WO 2010090645 A1 WO2010090645 A1 WO 2010090645A1
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WIPO (PCT)
Prior art keywords
populations
microbead
microbeads
fluorescent
intensity profiles
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Application number
PCT/US2009/033519
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English (en)
Inventor
Abraham Schwartz
Original Assignee
Abraham Schwartz
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abraham Schwartz filed Critical Abraham Schwartz
Priority to PCT/US2009/033519 priority Critical patent/WO2010090645A1/fr
Publication of WO2010090645A1 publication Critical patent/WO2010090645A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • G01N21/278—Constitution of standards
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2015/1014—

Definitions

  • the present invention relates to microbead populations, and more particularly to a method of producing microbead populations that mimic the fluorescence intensity profile distribution of fluorescent biological cells so that they may be used as a standard for flow cytometry.
  • Microbeads have been used as surrogate cell standards in various biological fields for many years. This is especially true since highly uniform microbeads, in the 2 - 15 micron diameter range, have been first produced by John Ugelstad.
  • polymeric microbeads As a particle surrogate, polymeric microbeads have high physical stability, as well as the ability to be labeled with the same fluorochromes and dyes as used to label biological cells.
  • a great deal of work has been addressed to make microbeads useful as fluorescent particle reference and quantitative standards. When labeled with such fluorochromes and dyes, these microbeads exhibit many of characteristics as labeled biological cells, as determined by instrumentation of at least one of flow cytometry.
  • fluorochrome labeled microbeads appear to have similar forward and side light scatter properties, as well as similar spectral and intensity fluorescent properties as biological cells.
  • fluorescence intensity distribution profile of microbeads and biological cells labeled with the same fluorochrome can appear vastly different.
  • human lymphocytes labeled with a fluorescently conjugated CD8 antibody has a fluorescence intensity distribution, as shown in FIG 1. This distribution is really the combined response of CD8 suppressor cells, the tall intense population on the right end of the distribution, and CD8 cyto-toxic cells, the low widely spread population to the left of the CD8 suppressor cells.
  • the present invention solves this need by providing a method to manipulate a population of microbeads labeled with a fluorescent material or optical dye so that it mimics the fluorescent intensity distribution of a biological cell population labeled with the same fluorescent material or dye.
  • This may be accomplished by a number of ways which include, but are not limited to at least one of: 1) mixing microbead populations of different intensities and uniformities, 2) manipulating the method of addition of the fluorescent material or 3) performing precision photo bleaching of a uniform population of microbeads.
  • the present invention provides method to manipulate a population of microbeads labeled with a fluorescent material or dye so that it mimics the fluorescent intensity distribution of a biological cell population labeled with the same fluorescent material or optical dye, by mixing different uniform populations of microbeads labeled with a fluorescent material in specific proportions, the overall fluorescent intensity pattern of the mixture can result in the same intensity pattern of the a cell population of interest labeled with the same material.
  • the intensity profile of a labeled cell population is also mimicked by carefully adjusting the physical parameters during the dying or polymerization procedure of the microbeads. Namely, adjusting the rate of dye addition, the stirring conditions and varying concentrations of the fluorescent material during the process.
  • a fluorescence intensity profile of a microbead population is accomplished using a process referred to as photo bleaching. That is, when fluorescent materials are exposed to strong light, especially wavelengths within their absorption spectra, the fluorescent molecules undergo destruction at some intrinsic rate, resulting in lower fluorescent intensity of the population as a whole. However, if different portions of the uniform population of microbeads are photobleached in a precise and predetermined manner at specific rates, the resulting intensity profile of the microbead population can be made to mimic the intensity profile of the labeled biological cells. This process is referred to as precision photo bleaching.
  • FIG. 1 shows a fluorescence intensity distribution of CD8 labeled with a fluorescent CD8 antibody.
  • FIG. 2 shows a fluorescence intensity distribution microbeads labeled with the same fluorochrome as bound to the CD8 antibody in Fig. 1.
  • FIG. 3 shows a plot of an intensity distribution from FIG. 9
  • FIG. 4 shows a plot of the inverted intensity distribution percentile from FIG.
  • FIG. 5 shows a plot of the sorted inverted intensity distribution percentile from
  • FIG. 6 shows the density gradient used to perform precision photo bleaching.
  • FIG. 7 shows the apparatus used to photo bleach a population of microbeads according to the present invention.
  • FIG. 8 shows fluorescence intensity profiles of a microbead population (a) before and (b) after photo bleaching through a optical density gradient.
  • FIG. 9 shows data from a list mode file indicating the number of events in each intensity channel of the histogram.
  • FIG. IO shows data from FIG. 9 that has been inverted.
  • FIG. 11 shows data from FIG. 10 that has been normalized to a density percentage.
  • FIG. 12 shows normalized data from FIG. 11 that has been sorted.
  • the preferred embodiment of the invention involves a method, an algorithm, and a precision photo bleaching apparatus for the production of microbeads that provide specific fluorescence intensity profiles to microbead populations that mimic fluorescently labeled biological cells.
  • the microbeads are uniformly spread over a given area. This may be done by filling a shallow flat-bottomed container and allowing the microbeads to settle uniformly onto the bottom of the container. An optical density gradient is placed over these microbeads and a light source of optimal wavelengths is directed through density gradient onto the uniform layer of microbead. The intensity and length of time the light is directed onto the microbeads controls the overall fluorescence intensity of the population, however, the intensity profile within the microbead population will retain an intensity profile across the illuminated area inversely proportional to the density gradient covering the microbeads.
  • the microbead surrogate cell standard is produced from a profile of an actual fluorescently labeled biological cell population by obtaining a list mode file from the cell population of interest with a flow cytometer.
  • the channel number and correlated event data from the list mode file is then transferred to a spreadsheet indicating the number of events represented in each channel of the intensity scale as shown in FIG. 9.
  • This intensity profile is then inverted by subtracting the number of events in each channel from the value in the channel with the maximum events as shown in FIG 10.
  • These inverted event values are then numerically sorted as shown in FIG. 11.
  • This sorted list is then converted to a percent of the maximum value as shown in FIG. 12.
  • These converted sorted numbers are then used to generate a density gradient as shown in FIG. 3, through which a uniform light source is directed onto a uniform layer of microbeads so that areas of the microbeads will photo bleach to a relative value inversely proportional to the density gradient covering them as shown in FIG. 6.
  • Example 2 Controlling fluorescence intensity distribution during microbead dying procedure.
  • propidum iodide dissolved in methanol was introduced at the top of the microbead suspension without stirring. The PI was allowed to diffused down through the suspension while the microbeads were settling to the bottom. After 30 minutes, the methanol was decanted from the microbeads and they were re- suspended in PBS containing 0.1% Tween 20. The fluorescence intensity distribution of the microbead population was found to have a wide distribution skewed to the left, >60%CV.
  • a highly uniform population of fluorescein-labeled microbeads was allowed to settle over an area so that the resulting microbeads formed a uniformly thick layer. Most of the suspension solution was removed so that the microbeads were covered by only a few millimeters of solution.
  • An optical density gradient produced by applying the algorithm to a list mode histogram file from gated CD8 FITC labeled lymphocytes. This gradient was placed 5 mm above the uniform layer of microbeads and a 5Ow high intensity lamp was directed onto the gradient for 2 hours so that the layer of microbeads was exposed to the resulting gradient light levels. The microbeads were then re-suspended and washed in PBS containing 0.1% Tween 20. The resulting fluorescent intensity histogram of the precision photobleached microbeads mimicked the gated CD8 FITC labeled lymphocytes

Abstract

L'invention concerne un procédé de production de populations de microbilles qui imitent la distribution de profils d'intensité de fluorescence de cellules biologiques fluorescentes, de telle sorte qu'elles peuvent être utilisées comme référence en cytométrie de flux.
PCT/US2009/033519 2009-02-09 2009-02-09 Nouvelle génération de microbilles fluorescentes de référence comme substitut cellulaire WO2010090645A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2009/033519 WO2010090645A1 (fr) 2009-02-09 2009-02-09 Nouvelle génération de microbilles fluorescentes de référence comme substitut cellulaire

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Application Number Priority Date Filing Date Title
PCT/US2009/033519 WO2010090645A1 (fr) 2009-02-09 2009-02-09 Nouvelle génération de microbilles fluorescentes de référence comme substitut cellulaire

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WO2010090645A1 true WO2010090645A1 (fr) 2010-08-12

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3631772A (en) * 1969-07-09 1972-01-04 Bell Telephone Labor Inc Method and apparatus for characterizing photoresist
US4714682A (en) * 1985-12-11 1987-12-22 Flow Cytometry Standards Corporation Fluorescent calibration microbeads simulating stained cells
US5084394A (en) * 1984-12-24 1992-01-28 Vogt Robert F Method for corrective calibration of a flow cytometry using a mixture of fluorescent microbeads and cells
US5380663A (en) * 1984-12-24 1995-01-10 Caribbean Microparticles Corporation Automated system for performance analysis and fluorescence quantitation of samples

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3631772A (en) * 1969-07-09 1972-01-04 Bell Telephone Labor Inc Method and apparatus for characterizing photoresist
US5084394A (en) * 1984-12-24 1992-01-28 Vogt Robert F Method for corrective calibration of a flow cytometry using a mixture of fluorescent microbeads and cells
US5380663A (en) * 1984-12-24 1995-01-10 Caribbean Microparticles Corporation Automated system for performance analysis and fluorescence quantitation of samples
US4714682A (en) * 1985-12-11 1987-12-22 Flow Cytometry Standards Corporation Fluorescent calibration microbeads simulating stained cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BRAECKMAN ET AL.: "Encoding microcarriers by spatial selective photobleaching", NATURE MATERIALS, vol. 2, March 2003 (2003-03-01), pages 169 - 173, XP002495700, DOI: doi:10.1038/nmat828 *

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