United States Patent [191
`Fulwyler
`
`[11]
`[45]
`
`Patent Number:
`Date of Patent:
`
`4,499,052
`Feb. 12, 1985
`
`[75]
`[73]
`
`121]
`[22]
`151]
`[52]
`
`[53]
`
`[56]
`
`Appl. No.:
`Filed:
`
`
`
`154] APPARATUS FOR DISTINGUISHING
`MULTIPLE SUBPOPULATIONS OF CELLS
`Mack J. Fulwyler, Sunnyvale, Calif.
`Inventor:
`Assignee:
`Becton, Dickinson and Company,
`Paramus, NJ.
`412,648
`Aug. 30, 1982
`G01N 21/64; G01N 33/50
`Int. Cl.3 US. Cl. ................................. .. 422/52; 250/4612;
`422/68; 436/63; 436/172
`Field of Search .................. .. 422/68, 52; 436/172,
`436/63; 250/4612; 356/36, 39
`References Cited
`U.S. PATENT DOCUMENTS
`3,497,690
`3,657,537
`3,684,377
`
`Wheeless, Jr. et al. ........ .. 250/4612
`Wheeless, Jr. et a1. ........ .. 250/4612
`Adams et a1.
`.. 250/4612 X
`
`Bonner et a1. . . . . .
`Stillman et a1.
`
`. . . . . .. 356/73 X
`.. 250/4612 X
`
`2/1970
`4/1972
`8/1972
`7/1974
`2/1975
`10/1975
`lO/l975
`
`Fulwyler ...... ..
`
`..
`
`.
`
`Kleinerman .................... .. 250/4612
`
`3:916:05
`OTHER PUBLICATIONS
`Latt et al., Journal of Histochemistry and Cytochemis
`try, vol. 27, No. 1 pp. 65-71, 1979.
`
`Clausen et al., International J. Andrology Suppl. 2 (2)
`1978, pp. 513-522.
`
`Primary Examiner-Arn0ld Turk
`Attorney, Agent, or Firm-Richard J. Rodrick
`[57]
`ABSTRACT
`A method of distinguishing multiple subpopulations of
`cells from a single sample of cells of a variety of types
`comprises labeling particles with two or more marking
`agents. These particles are marked in a plurality of
`different pre~selected ratios of the agents ranging be
`tween zero percent and one hundred percent of each
`agent. Each such agent has distinguishing, quanti?able
`marking characteristics. The differently labeled parti
`cles are mixed with cells suspected of having speci?c
`receptors for the differently labeled particles. Each cell
`is analyzed to determine the ratio of any two identi?able
`marking characteristics associated with each cell so that
`it can be classi?ed in a subpopulation category if its
`ratio of marking characteristics is related to one of the
`pre-selected ratios of marking agents.
`An apparatus for carrying out the above-described
`method is also within the purview of the present inven
`tion.
`
`10 Claims, 2 Drawing Figures
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`APPARATUS FOR DISTINGUISHING MULTIPLE
`SUBPOPULATIONS OF CELLS
`
`5
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The present invention relates to a method and appara
`tus for distinguishing multiple subpopulations of parti
`cles, and more particularly, concerns a method and
`apparatus for simultaneously distinguishing and enua
`merating multiple subpopulations of cells which have
`been labeled with different ?uorochromes.
`2. Description of the Prior Art
`Presently known and available ?ow-through cytome
`ters and the like particular detecting devices commonly
`include two channels for the detection of two subpopu
`lations of cells in a mixture. For example, devices are
`known which include two ?uorescence channels which
`can detect cells speci?cally labeled with two ?uores
`cent agents associated with the respective ?uorescence
`channels. In these type devices, a complete fluorescence
`channel including the electrical circuitry and ?uores
`cence detectors has been required for each ?uoro
`chrome-treated cell to be detected in the mixture of
`cells in the sample being analyzed. Therefore, in order
`25
`to detect multiple subpopulations of cells in a sample‘
`using ?ow-through cytometry, an equivalent number of
`fluorescence channels is required using the known,
`conventional devices. A further limitation is that the
`nature of excitation and emission characteristics of
`?uorochromes makes it dif?cult to acquire more than
`two ?uorochromes, attachable to protein, which pro
`vide emissions suf?ciently separated in wavelength.
`Some representative devices using conventional ?ow
`through cytometry are described in US. Pat. Nos.
`4,198,160; 3,738,759; 3,864,571; and in “A Proposal for
`an Automatic Multi-parameter Analyzer for Cells
`(AMAC),” by Robert C. Leif, Automated Cell Identi?
`cation and Cell Sorting, edited by George L. Wied, Aca
`demic Press, New York 1970, pages 131-159.
`There are many instances when it is desirable to be
`able to detect multiple subpopulations of cells from a
`sample mixture. However, as alluded to above, one of
`the disadvantages found in conventional equipment is
`that a plurality of ?uorochromes would have to be
`employed for labeling the cells, as well as an equivalent
`number of ?uorescence channels to monitor the speci?c
`spectral characteristics associated with the individual
`?uorochromes. Moreover, a suf?cient plurality of
`?uorochromes is not presently available. Clearly, this
`has created formidable problems. While it is desirable to
`be able to detect, and also enumerate, multiple subpopu
`lations of cells from a sample mixture, it is even more
`desirable to minimize the number of ?uorochromes
`employed as well as the number of ?uorescence chan
`nels and the associated circuitry. With this in mind, the
`present invention is directed to solving the aforemen
`tioned problem, while satisfying the desired need for the
`determination of multiple subpopulations of cells from a
`sample mixture.
`
`55
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`35
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`45
`
`SUMMARY OF THE INVENTION
`A method of distinguishing multiple subpopulations
`of particles from a single sample of particles of a variety
`of types includes labeling receptive substances with two
`65
`or more marking agents in a plurality of different pre
`selected ratios of said agents. The ratios may range
`between zero percent and one hundred percent of each
`
`1
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`4,499,052
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`2
`agent which has a distinguishing and quanti?able mark
`ing characteristic. This method further includes mixing
`the differently labeled substances with particles sus
`pected of having speci?c receptors for the differently
`labeled substances. Each particle is analyzed to deter
`mine the ratio of the two identifiable marking character
`istics associated with each particle. Thereafter, each
`particle can be classi?ed in a subpopulation category if
`its ratio of marking characteristics is related to one of
`the pre-selected ratios of marking agents.
`In a preferred embodiment of this aspect of the pres
`ent invention, the method includes labeling antibody
`proteins with two ?uorochromes in a plurality of differ
`ent pre-selected ratios. Each ?uorochrome has distinct
`emission spectra. Excitation energy is provided to the
`cells by ?ow-through cytometry techniques to thereby
`excite both types of the ?uorochromes. Each cell is
`analyzed to determine the fluorescence emitted by both
`excited ?uorochromes to thereby establish the ratio of
`the ?uorescence emissions. Thereafter, each cell is clas
`si?ed by subpopulation category if related to one of the
`pre-selected ratios of labeled antibody proteins. Simul
`taneous enumeration of the cell subpopulations can also
`be achieved with the present invention.
`Another aspect of the present invention is an appara
`tus for distinguishing multiple subpopulations of parti
`cles from a sample of particles ?owing in a liquid path.
`The particles have been labeled with up to two or more
`different marking agents having distinguishing and
`quanti?able characteristics. The apparatus includes
`means for separately detecting the quanti?able charac
`teristics associated with each particle and determining a
`ratio of any two quanti?able characteristics thereof.
`Means for recording the ratios is provided so that the
`particles can be classi?ed into a plurality of subpopula
`tion categories.
`In a preferred embodiment of this aspect of the pres
`ent invention, the apparatus simultaneously distin
`guishes and enumerates multiple subpopulations of cells
`which have been labeled with up to two or more differ
`ent ?uorochromes. Means for exciting ?uorochromes
`on each cell as it ?ows in a liquid path is provided. This
`preferred apparatus further includes means for sepa
`rately detecting the quantity of fluorescence emitted by
`the two different ?uorochromes associated with each
`cell and determining the ratio of fluorescence quantities
`of the two ?uorochromes. Further, there is means for
`displaying the ratios so that the cells can be classi?ed
`into a plurality of subpopulation categories and enumer
`ated.
`It is also within the purview of‘ the present invention
`to determine ratios of ?uorescenated particles having
`similar emission characteristics, but different excitation
`characteristics. Different light sources for excitation
`might be required, while only one ?uorescence detector
`need be employed. Also, ratios may be determined in
`accordance with the present invention utilizing ?uore
`scenated particles having both different excitation and
`emission characteristics.
`In accordance with the principles of the present in
`vention, a number of advantages and objectives are
`attained. Primarily, the present invention permits the
`analysis and determination of multiple subpopulations
`of particles or cells in a greater quantity than the num
`ber of ?uorochromes employed. Further, a greater
`number of cell subpopulations can be determined than
`the number of ?uorescence detection channels, and
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`associated electronic circuitry, utilized. In the present
`invention, a straightforwardly constructed instrument
`needs only two ?uorescence channels that are capable
`of detecting distinct emission spectra of two different
`fluorochromes. On analysis in an apparatus as described
`above, cell subpopulations are distinguished by deter
`mining the ratio of the two distinct fluorochromes asso
`ciated with each cell using only two ?uorescence chan
`nels, each directed to detecting the distinct emission
`spectra of the fluorochromes. By using a ratio, many
`subpopulations of cells labeled with only two distin
`guishable fluorochromes or other marking agents can
`be determined. Moreover, in the flow-through cytome
`try techniques envisaged by the present invention, mul
`tiple cell subpopulations can be detected in rapid order
`from a single sample of cells. The present invention not
`only provides for the detection of multiple subpopula
`tions of cells, but also provides for the simultaneous
`enumeration of the cells so detected. Furthermore, by
`reliance upon a ratio of signals detected with respect to
`each cell or particle, they are distinguished by the ratio
`parameter which is independent of the quantity of ?uo
`rescence marking agents bound to a cell; in addition,
`cell subpopulation distributions do not overlap each
`other to cause erroneous or inaccurate results. An addi
`tional advantage is that it is possible to detect the non
`speci?c binding of fluorescenated agents to particles.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a schematic representation of a cytometric
`apparatus for detecting two ?uorescence characteristics
`of individual particles moving in a flow path from the
`sample source; and
`FIG. 2 is a graphic representation of multiple subpop
`ulations of particles determined by a ratio distinction
`technique in accordance with the principles of the pres
`ent invention.
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`antibodies can be mixed together and reacted with a cell
`population in a sample mixture. Each antibody, with a
`known ratio of fluorochromes attached thereto, will
`then bind to those cells having speci?c receptors there
`for, thereby labeling subpopulations of cells. Once this
`treatment has been completed and speci?c cell subpop
`ulations labeled, the cell sample is placed in a sample
`source 12 associated with detection apparatus 10 as seen
`in FIG. 1.
`Before explaining the operation of detection appara
`tus 10, in general terms the two fluorochromes, or other
`marking agents if so used in conjunction with this inven
`tion, may be applied to the receptive substances such as
`antibody proteins in different pre-selected ratios. These
`ratios range between zero percent and one hundred
`percent of each fluorochrome; i.e., there may be no
`fluorochrome of the ?rst type on an antibody protein,
`while there is one hundred percent of a fluorochrome of
`the second type on that same antibody protein. Of
`course, various ratios of the two fluorochrornes lying
`between the extremes of zero percent and one hundred
`percent fall within the purview of the present invention.
`Furthermore, present cytometric techniques and equip
`ment used therefor should allow the detection of at least
`?ve different cell subpopulations using the method and
`apparatus as described herein. It is understood, how
`ever, that more than ?ve cell subpopulations may be
`distinguished by the present method and apparatus, but
`the quality of the signal may not be as strong for more
`than ?ve ratio measurements. Further, the use of three
`or more fluorochromes significantly increases the num
`ber of distinguishable ratios which are possible with the
`present invention.
`Turning now to the speci?cs of detection apparatus
`10 in FIG. 1, sample source 12 contains the substances,
`such as cells, which have been treated with different
`marking agents, such as fluorochromes, in a plurality of
`different preselected ratios. For the ensuing discussion,
`two such fluorochromes are employed to treat the cells,
`merely for exemplary and descriptive purposes. The
`treated cells 14 are delivered in a fluid stream, prefera
`bly individually, to and through sensing region 15, such
`as an ori?ce, which will allow the optical aspects of the
`cells to be detected. Sensing features are well-known in
`flow-through cytometric devices, and one such sensing
`arrangement is disclosed in an article by Thomas, R. A.,
`et al. “Combined Optical and Electronic Analysis of
`Cells with the AMAC Transducers,” The Journal of
`Histochemistry and Cytochemistry, Vol. 25, No. 7,
`pages 827-835, 1977. As each treated cell 14 passes
`through sensing region 15, light from a light source 16
`is directed at the cells. Light source 16 delivers light to
`the cells and may include lasers, Mercury or Xenon are
`lamps, or the like, capable of emitting a number of lines
`through a wide range of color regions. Also, the light
`from light source 16 in the embodiment being described
`should be suf?cient to cause excitation of the two differ
`ent fluorochromes used to treat cells 14. Thus, when the
`light strikes each cell 14 the ?uorochromes bound
`thereto become excited thereby providing a mechanism
`for distinguishing the fluorescence characteristics of
`each cell. It is appreciated that when fluorochromes are
`selected having different excitation ranges, it may be
`necessary to employ more than one light source to
`cover the disparate wavelengths of excitation.
`As each cell passes through the sensing region it is
`then collected in a receptacle 18; although not shown
`herein, the cells could be sorted according to known
`
`DETAILED DESCRIPTION
`While this invention is satis?ed by embodiments in
`many different forms, there is shown in the drawings
`and will herein be described in detail a preferred em
`bodiment of the invention, with the understanding that
`the present disclosure is to be considered as exemplary
`of the principles of the invention and is not intended to
`limit the invention to the embodiment illustrated. The
`scope of the invention will be measured by the ap
`pended claims and their equivalents.
`Referring to the drawings, and FIG. 1 in particular,
`there is illustrated a schematic representation of a cy
`tometric apparatus 10 for detecting cells, or other parti
`cles, having particular parameters. Before any particles
`are analyzed in detection apparatus 10, they are treated
`with a plurality of marking agents that have quanti?able
`marking characteristics, preferably different from each
`other. For example, in tests wherein cells are to be
`classi?ed, it is most advantageous to work with anti
`body proteins. In general, these antibody proteins are
`labeled with two marking agents, preferably ?uoro
`chromes, although three or more such agents may be
`utilized. Each fluorochrome has distinct emission and
`/or excitation spectra in speci?cally de?ned color
`bands. The fluorochromes are bound to antibody prote
`ins such that the number of these proteins labeled with
`each fluorochrome form a known ratio. By labeling
`different antibody proteins, each being speci?c for re
`ceptors on a certain cell type, with different ratios of
`fluorochromes, a plurality of these differently labeled
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`sorting techniques wherein subpopulations of cells can
`be collected separately. Fluorescent light from each
`cell, including ?uorescence from up to two ?uoro
`chromes having distinct excitation spectra, is then di
`rected to a dichroic mirror 19. The purpose of dichroic
`mirror 19 is to separate two different colors along the
`re-radiated light path generated by the ?uorescence
`characteristics of each cell. In this fashion, the two
`different colors can be analyzed separately to thereby
`form a ratio as hereinafter described. Dichroic mirror
`19 would be selected to separate, for example, the green
`from the red regions of the color spectrum. Wave
`lengths in the ?rst color region would be re?ected
`along optical path 20, while wavelengths of the second
`color region would be transmitted through dichroic
`mirror 19 along optical path 21. Each light path either
`reflected or transmitted through the dichroic mirror is
`then detected by ?uorescence detectors 22 and 24, re
`spectively, provided to receive the light energy sepa
`rated into the two regions. Fluorescence detectors 22
`and 24 may be conventional photomultiplier tubes
`which convert optical signals into electrical signals.
`These electrical signals are then fed to respective pulse
`processing electronics 25 and 26 wherein the electrical
`signals are processed for analysis purposes.
`As part of this analysis, and preferably as part of the
`electronics of the apparatus herein described, a ratio of
`the electrical signals is determined. In the ratio deter
`mining means 27 the ?uorescence signal from fluores
`cence detector 22 is related as a ratio to the fluorescence
`signal from ?uorescence detector 24, or vice versa.
`Ratio means 27 thereby provides a mechanism to deter
`mine the ?uorescence emitted by both excited ?uoro
`chromes associated with each cell and to establish the
`ratio of their ?uorescence emissions. This ratio informa
`tion is then fed to display means 28. The combination of
`the electronics 25 and 26, ratio means 27 and display
`means 28 are all preferably electrical circuits which will
`provide for various displays, information presentation,
`accumulation or recordation of the ratio of ?uorescence
`signals associated with each cell being analyzed. The
`electrical components to provide analysis of the electri
`cal signal relating to ?uorescence may include state of
`the art technology and may vary according to the level
`of sophistication of the analysis and data presentation.
`One such electrical system for ?uorescence determina
`’ tions is described in US. Pat. No. 3,826,364.
`Display means 28 preferably includes a screen to
`visually observe in graphic form the classi?cation of
`each cell by subpopulation category. In addition, appa
`ratus 10, along with the electronics and display may be
`designed to pre-program ratio information into the cir~
`cuitry. For example, and referring now to FIG. 2, the
`electronics and display can be pre-programmed to in
`clude speci?cally de?ned ?uorescence ratios along the
`X-axis of the screen. These ratios would include the
`same ratios of ?uorochromes pre-selected to treat the
`antibody proteins which are speci?c for certain cell
`types. The Y-axis of the screen can be pre-programmed
`to plot the number of cells associated with the speci?
`cally de?ned ratios along the X-axis. In this fashion, a
`graphic, histogram representation of the subpopulations
`of cells classi?ed into speci?c categories can be visual
`ized and, if desired, recorded. As can be seen in FIG. 2,
`?ve subpopulations of cells have been identi?ed having
`speci?c ratios of ?uorescence, i.e., <l/l0, l/3, l/l,
`3/1, and > 10/ 1. The area under each of the cell type
`peaks, A to E, would provide the number of cells of that
`
`6
`type measured. If desired, the electronics of this appara
`tus could be designed to calculate the approximate num
`ber of cells classi?ed into each subpopulation.
`It is appreciated that the present apparatus therefore
`provides for the classi?cation of cell subpopulations and
`the numbers of cells in each subpopulation as a simulta
`neous determination, which can then be displayed to the
`operator. Moreover, because a ratio is used, cells are
`distinguished by this ratio technique independent of the
`quantity of ?uorochrome-treated antibodies bound to a
`cell; as can be seen in FIG. 2, cell subpopulation distri
`butions do not overlap because of the normalizing effect
`of the ratio. In making these classi?cations of cell sub
`populations, windows 30 are provided around each
`pre-selected ratio which form a range for making the
`speci?c classi?cations. Upon analysis, the cells which
`fall within windows 30 on the ratio scale are taken to be
`speci?cally labeled; cells to which ?uorochromes or
`other marking agents are non-speci?cally bound would
`provide ratios outside of the permitted windows, such
`as in the valley regions 31 between the peaks of the
`curve as seen in the graphic representation of FIG. 2.
`Accordingly, cells outside of windows 30, in valley
`regions 31, are taken to be non-speci?cally labeled and
`would be rejected electronically. However, non-speci?
`cally labeled cells provide ratios lying outside of the
`permitted ratio windows enabling separate enumeration
`of these cells.
`Whereas FIG. 2, and the apparatus in general being
`described, distinguishes and classi?es ?ve different cell
`subpopulations, the number of cell types distinguishable
`by the method and apparatus of this invention may
`exceed ?ve. However, signal strength should be ade
`quate to resolve closer ratios, i.e., 9/1, 8/1, 7/1, etc. It
`should also be understood that the present method is
`most ef?cient when there is no cross reaction, i.e., each
`antibody protein labels only one cell type and each cell
`type accepts, for binding purposes, only one antibody
`protein type.
`For illustrative purposes of the present invention the
`following examples exemplify, but do not limit the
`scope of, the mechanism for detecting and distinguish
`ing multiple subpopulation of particles:
`EXAMPLE 1
`A ?uorescent polymer is synthesized having pre
`selectable proportions of two ?uorochromatic mono
`mers, in this case, ?uorescein and rhodamine. Fluores
`cein will emit ?uorescence when excited in the blue
`color region; on the other hand, rhodamine will emit
`?uorescense when excited in the yellow color region.
`Five polymer preparations are synthesized as follows:
`Polymer Preparation 1-—100% ?uorescein, 0% rho
`damine.
`Polymer Preparation 2—75% fluorescein, 25% rho
`damine.
`Polymer Preparation 3—50% fluorescein, 50% rho
`damine.
`Polymer Preparation 4—25% fluorescein, 75% rho
`damine.
`Polymer Preparation 5-—0% ?uorescein, 100% rho
`damine.
`Antibody proteins which are speci?c for a certain
`cell type, herein designated as cell type A, are then
`labeled with Polymer Preparation 1; antibody proteins
`speci?c for cell type B are labeled. with Polymer Prepa
`ration 2; antibody proteins speci?c for cell type C are
`labeled with Polymer Preparation 3; antibody proteins
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`speci?c for cell type D are labeled with Polymer Prepa
`ration 4; and antibody proteins speci?c for cell type E
`are labeled with Polymer Preparation 5.
`All of the conjugated (labeled) antibodies are mixed,
`and then the mixture is added to a cell sample. The cell
`sample includes cells believed to have speci?c receptors
`for the differently labeled antibodies. A—type cells
`would then be labeled with only the fluorochromes of
`Polymer Preparation 1, B-type cells with Polymer
`Preparation 2, C-type cells with Polymer Preparation 3,
`D-type cells with Polymer Preparation 4 and E-type
`cells with Polymer Preparation 5.
`On analysis in an apparatus such as described in FIG.
`1, each treated cell is analyzed and its blue fluorescein
`signal and yellow rhodamine signal are electrically
`detected and formed into a ratio as described in con
`junction with apparatus 10 above. For A~type cells this
`ratio is greater than 10/1; for B-type cells, the ratio is
`3/ l; for C-type cells the ratio is 1/ l; for D-type cells the
`ratio is l/3 and for E-type cells the ratio is <l/10.
`Thus, by determining the yellow/blue fluorescence
`ratio of each cell as it passes through the detection
`apparatus, it can be classi?ed as belonging to one of the
`?ve cell types. A graphic representation of this classi?
`cation is similar to that illustrated in FIG. 2.
`
`25
`
`EXAMPLE 2
`Two polymer preparations are employed, one con
`taining fluorescein only and another containing rhoda
`mine only. The following preparations are prepared:
`Preparation l—lOO% of antibody protein is labeled
`with polymer containing ?uorescein.
`Preparation 2—75% of antibody protein is labeled
`with polymer containing fluorescein, and 25% of the
`antibody protein is labeled with polymer containing
`rhodamine.
`Preparation 3-—50% of antibody protein is labeled
`with polymer containing ?uorescein, and 50% of anti
`body protein is labeled with polymer containing rhoda
`mine.
`Preparation 4—25% of antibody protein is labeled
`with polymer containing tluorescein, and 75% of anti
`body protein is labeled with polymer containing rhoda
`mine.
`Preparation 5-—100% of antibody protein is labeled
`with polymer containing rhodamine.
`, When a mixture of these ?ve antibody preparations is
`added to a mixed cell population (suspected of having
`speci?c receptors for the differently labeled antibody
`proteins), A-type cells accept only antibodies labeled
`with Preparation 1; B-type cells accept antibodies la
`beled with Preparation 2; C-type cells accept antibodies
`labeled with Preparation 3; D-type cells accept antibod
`ies labeled with Preparation 4 and E-type cells accept
`antibodies labeled with Preparation 5. Upon analysis in
`a flow-through cytometer such as the apparatus of FIG.
`1, data obtained is similar to that shown in Example 1.
`
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`55
`
`EXAMPLE 3
`The preparations of Example 2 are repeated, except
`that conventional FITC (fluorescein isothiocyanate)
`and RITC (rhodamine isothiocyanate) labeled antibod
`ies are used in place of the ?uorescein-containing and
`rhodamine-containing polymers. Results of analyzing
`these cells in a ?ow-through, dual fluorescence cytome
`ter, would be substantially similar to the results shown
`in Example 1.
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`4,499,052
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`8
`EXAMPLE 4
`Microspheres are produced which contain prese
`lected ratios of two fluorochromes having different
`emission characteristics. Microspheres may be pro
`duced in accordance with U.S. Pat. No. 3,790,492. The
`microsphere preparations are then substituted for the
`polymer preparations of Example 1. On flow-through
`analysis, the data obtained is similar to that shown in
`Example 1.
`
`EXAMPLE 5 _
`Microspheres similar to those in Example 4 are pre
`pared in which fluorescein-containing microspheres
`and rhodamine-containing microspheres are substituted
`for the two types of polymers listed in Example 2. Upon
`analysis in a flow—through cytometer, the data obtained
`is substantially similar to that shown in Example 1.
`‘Thus, the present invention provides a method and
`apparatus for detecting and distinguishing multiple sub
`populations of particles in a larger particle population.
`Advantageously, many more subpopulations may be
`distinguished than the number of fluorescence agents
`and ?uorescence channels employed in this invention.
`By utilizing a ratio of ?uorescence signals, particle sub
`populations can be detected and classi?ed, while at the
`same time enumerating the number of particles classi
`?ed into each particle subpopulation.
`What is claimed is:
`1. An apparatus for distinguishing multiple subpopu
`lations of particles, each of which have been labeled
`with different fluorochromes in pre-determined ratios
`comprising: means for exciting the fluorochromes on
`each particle as it flows in a liquid path; means for de
`tecting the quantity of fluorescence emitted by the dif
`ferent fluorochromes associated with each particle and
`determining the ratio of fluorescence quantities of any
`two fluorochromes associated with each particle; and
`means for classifying said particles into a plurality of
`subpopulations categories by associating said deter
`mined ratios with pre-determined ratios.
`2. The apparatus of claim 1 wherein the means for
`exciting includes a source of light and the means for
`detecting includes photodetector devices each adapted
`to detect light energy in speci?cally de?ned color re
`gions.
`3. The apparatus of claim 1 which further includes
`means to simultaneously detect the quantity of fluores
`cence emitted by different ?uorochromes associated
`with each particle.
`4. The apparatus of claim 1 wherein the ratios of
`fluorescence quantities are determined by electrical
`determination means and further including means for
`feeding said determined ?uorscence quantities to the
`means for classifying.
`5. The apparatus of claim 4 wherein said electrical
`means includes means for creating said subpopulation.
`categories based on separately identi?able ratios which
`have been predetermined to coincide with the pre
`determined ratios of fluorochromes on said particles.
`6. An apparatus for distinguishing multiple subpopu
`lations of particles from a sample of cells flowing in a
`liquid path, said particles having been labeled in pre
`determined ratios with different marking agents having
`quanti?able characteristics, comprising: means for de
`tecting the quanti?able characteristics associated with
`each particle and determining a ratio of any two quanti
`?able characteristics associated with each particle; and
`
`Luminex Ex. 1006
`Luminex/Irori - Page 7
`
`

`

`4,499,052
`
`9
`means for associating said determined ratios with pre
`determined ratios so that the particles can be classi?ed
`into a plurality of subpopulation categories.
`7. The apparatus of claims 1 or 6 which further in
`cludes means to determine the approximate number of
`particles classi?ed into each subpopulation simulta
`‘neously with the detection of particle subpopulation
`categories.
`8. The apparatus of claim 7 which further includes
`means to determine the approximate number of parti
`
`10
`cles which are non-speci?cally labeled and which do
`not fall within any de?ned subpopulation.
`9. The apparatus of claim 8 wherein said particle
`number determining means includes display means for
`indicating the number of particles classi?ed within or
`without of each subpopulation‘
`10. The apparatus of claim 9 wherein said display
`means includes means for graphically presenting said
`subpopulation categories based on separately identi?a
`ble ratios which have been pre-determined to coincide
`with the predetermined ratios of ?uorochromes on said
`particles.
`
`*
`
`*
`
`*
`
`*
`
`>|=
`
`20
`
`25
`
`35
`
`45
`
`55
`
`60
`
`65
`
`Luminex Ex. 1006
`Luminex/Irori - Page 8
`
`