EP0678195A4 - Method of using preserved control cells in the calibration of fluorescent and light scatter measurements. - Google Patents

Method of using preserved control cells in the calibration of fluorescent and light scatter measurements.

Info

Publication number
EP0678195A4
EP0678195A4 EP94906567A EP94906567A EP0678195A4 EP 0678195 A4 EP0678195 A4 EP 0678195A4 EP 94906567 A EP94906567 A EP 94906567A EP 94906567 A EP94906567 A EP 94906567A EP 0678195 A4 EP0678195 A4 EP 0678195A4
Authority
EP
European Patent Office
Prior art keywords
cells
labelled
fluorescent
dye
assay
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP94906567A
Other languages
German (de)
French (fr)
Other versions
EP0678195A1 (en
Inventor
John A Maples
Ravinder K Gupta
Marcia Johnson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Coulter Corp
Original Assignee
Coulter Corp
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 Coulter Corp filed Critical Coulter Corp
Publication of EP0678195A1 publication Critical patent/EP0678195A1/en
Publication of EP0678195A4 publication Critical patent/EP0678195A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • G01N33/533Production of labelled immunochemicals with fluorescent label
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/1012Calibrating particle analysers; References therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Optical investigation techniques, e.g. flow cytometry

Definitions

  • the invention relates to the use of labelled preserved cells as controls to make compensation adjustments for the overlapping wavelength emissions of multiple fluorescent substances used as labels in multiple color biological analyses by flow cytometry.
  • the invention also describes the preparation of preserved labelled cells which have stability and shelf life characteristics not previously realized.
  • Control cells are essential for the accuracy and precision of clinical tests and immunoassays. In assays which use light scatter and fluorescent labels to identify cells and/or cellular components, control cells are used to form the basis for making compensation adjustments for areas of fluorescent overlap. The use of control cells and the making of the compensation adjustments is necessary to insure the reliability and accuracy of the test equipment and methods, and to insure reproducibility through time and from 1aboratory-to- laboratory.
  • cells and/or cellular substances are determined on the basis of either direct interaction with the fluorescent substance or indirect interaction via bonding of the fluorescent substance to a biological substance, for example, a monoclonal antibody, plant lectin and other substances which may be involved in the reaction with the cells or cellular components.
  • a biological substance for example, a monoclonal antibody, plant lectin and other substances which may be involved in the reaction with the cells or cellular components.
  • the problem is particularly important in flow cyto etry where the objective is to label cells with at least two distinct fluorochro es which reflect discrete cellular components by detecting a signal proportional to each fluorochrome and unbiased by any contribution from the other fluorochrome. Unless adjustments are made for the overlapping wavelengths, false "positive" readings may occur. It is not possible to choose wavelength filters which will optimally transmit the light of one overlapping] fluorochrome and totally block the light of the other fluorochrome, thereby avoiding the false positive readings. Most multiple color analytical systems provide "compensation" to eliminate the false positive readings.
  • Compensation is an electronic means of computing and subtracting overlapping fluorescent signals, i.e., the overlap contribution from a second fluorochrome is subtracted from the fluorescence reading of a first fluorescent label on the cells being analyzed.
  • the process is reversed and the overlap contribution from the first fluorochrome is subtracted from the fluorescence reading of the second fluorescent label.
  • there must be some method of determining whether too much or too little signal is subtracted relative to the specific fluorochrome and the targeted cells or cellular components.
  • Another consideration in flow cytometry is that is important that the size, shape and structure of the labelled calibration substance have a direct relationship to the size, shape and structure of the cells or cellular components being analyzed. For example, a large labelled spherical calibration or control particle would not be a proper match for an unknown consisting of small rectangular particles. .For this reason, biological cells are preferred over latex beads as controls.
  • This invention provides a method for using preserved, labelled biological cells as control cells for light scatter measurements and fluorescent calibration.
  • a provision providing for such cells enables the analyst to select the proper cells for making compensation adjustments and provides the ability to establish long term baselines for the comparison of samples analyzed at different points in time.
  • the invention describes a method of using labelled reconstituted preserved cells as control cells to calibrate light scatter and fluorescent intensity measurements in multiple color assays using two or more fluorescent labels and to provide for compensation adjustments in such assays which eliminate false positive readings caused by the overlapping wavelengths of different fluorescent substances.
  • the compensation factors used in the adjustments are determined electron ⁇ ically by the analytical instrument. The compensation factors prevent the appearance of false positive readings during the assay.
  • the assays with which the method is used are biological cells assays.
  • the method is an improvement over conventional techniques which require fresh cells or which use polymeric beads.
  • the use of labelled preserved cells according to the invention enables the analyst to establish a long term baseline for the comparison of samples assayed at different points in time.
  • the invention also describes preferred labelled preserved cells for use as the control cells and a method of preparing the same.
  • FIG 1A illustrates the gating, with no compensation adjustments, of the preserved cells on the basis of forward light scatter (FS) and log side light scatter.
  • FIG. IB illustrates false positive fluorescent staining of cells in Quad 2 due to lack of fluorescent compensation adjustments.
  • FIG. 1C further illustrates false positive fluorescent staining in Quad 2 due to lack of compensation adjustments.
  • FIG. ID illustrates that even without compensation adjustments there is no overlap region for FITC and ECD fluorescent emissions.
  • FIG. 2A illustrates the gating, with compensation adjustments of the preserved cells on the basis of forward light scatter and log side light scatter.
  • FIG. 2B illustrates the correct fluorescent reading obtained after sitting the appropriate compensation for overlapping fluorescent emissions.
  • FIG. 2C illustrates that with the appropriate fluorescent compensation for overlapping emission wavelengths, there are no false positive readings in Quads 1 and 2.
  • FIG. 2D further demonstrates that with the appropriate fluorescent compensation, these are no false positive readings in Quads 1 and 2.
  • the invention describes a method which uses pre ⁇ served cells to determine light scatter and compensation adjustments for wavelength emissions of multiple fluorescent substances used as labels in multiple color biological analysis.
  • any preserved, labelled cells which retain their authentic structural and antigenicity characteristics can be used according to the invention.
  • Lyophilized cells may be prepared according to U.S. Patent No. 5,059,518 issued October 29, 1991 for STABILIZED LYOPHILIZED MAMMALIAN CELL AND METHOD OF MAKING SAME.
  • the teachings of Patent No. 5,059,518 are incorporated herein by reference.
  • Other methods of preserving cells, bacteria, and other biological substance may be found in U.S. Patent Nos.
  • the labels used in the invention are fluorescent substances.
  • the labelling may be done by direct reaction between the label and biological cells or cellular components.
  • the label may be attached to (1) a reactive biological material, such as, a monoclonal antibody or (2) a reactive non-biological organic substance, for example, a carboxylic acid chloride, prior to attachment to the cells or cellular components.
  • fluorescent labels may be attached to a selective reactant substance such as a monoclonal antibodies and the labelled monoclonal antibodies then conjugated to cells.
  • the preferred method is to attach the label to a selectively reactive substance, and most preferably, to a monoclonal antibody.
  • the fluorescent labels may be selected from fluoro- scein, fluoroscein isothiocyanate (FITC), rhodamine, tetramethylenerhodamine isothiocyanate (TRITC), sulfor- hodamine 101 acid chloride (Texas Red), phycoerythrin (PE), allophycocyanin, phycoerythrin-Texas Red (PETR), 4-methylumbelliferone and other fluorescent substances known or found useful in the analysis of biological substances.
  • the conjugation of the label to the monoclonal antibody can be performed by any suitable and known method including direct reaction between the monoclonal antibody and the label or the use of bridging groups to connect the monoclonal antibody and the label.
  • the labelled lyophilized cells used herein as control cells may be prepared by reconstituting cells lyophilized according to Patent No. 5,059,518 and incubating the reconstituted cells with either a polyclonal or monoclonal antibody. Monoclonal antibodies specific to an antigenic site present on the cells are preferred. The antibody may be labelled before incubation with the reconstituted cells or it may be labelled after it has been attached to the reconstituted cells. The former is preferred. The resulting labelled cells are then used according to the claimed invention.
  • fresh cells may be labelled, for example, by incubating the fresh cells with an antibody.
  • the antibody may be labelled before or after incubation. Again, the former is preferred.
  • the cells are lyophilized according to said Patent No. 5,059,518. It has been determined that preservation by the method of said patent does not destroy or decrease the fluorochrome's fluorescent ability as is known to occur using other methods of preservation. Prior to their use as control cells according to the herein invention, the lyophilized labelled cells are reconstituted.
  • Directly labelled cells are those in which the label is attached to the cell without the use of an intervening antibody or other substance.
  • An anti-CD8 monoclonal was labelled with phycoerythrin (RD1, obtainable from Coulter Corporation of Miami, Florida) to produce a labelled antibody designated as LFL2.
  • An anti-CD4 monoclonal antibody was labelled with fluoroscein isothiocyanate (FITC) to produce a species designated as LFL1.
  • FITC fluoroscein isothiocyanate
  • a negative control antibody IgG b
  • ECD energy coupled dye, Coulter Corporation, Miami, Florida
  • FIG. 1A illustrates the gating of the preserved cells on the basis of forward light scatter (FS) and log side light scatter (LSS) included in electronic gate (1).
  • FS forward light scatter
  • LLS log side light scatter
  • IB illustrates the false positive fluorescent staining of cells in Quadrant (Quad) 2 due to the lack of fluorescence compensation, such that, there appears CD8-FITC positive cells in Quadrant 1 (LFL2+); CD4-RD1 positive cells in Quadrant 4 (LFL1+); LFLl(-) LFL2(-) cells in Quadrant 3; and LFL1(+) LFL2(+) cells in Quadrant 2.
  • the dual positive cells seen in Quadrant 2 are the result of the FITC emission wavelength overlapping into LFL2 (overlap between FITC and RD1 emissions) .
  • FIG. 1C illustrates the false positive fluorescence is due to the fact that there is no fluorescence compensation for overlapping wavelengths (refer to FIG. IB).
  • the dual positive cells seen in Quadrant 2 are the result of the emission wavelength from LFL2 (RD1 emission) overlapping with LFL3 (ECD emission).
  • FIG. ID illustrates that without compensation, the separate emissions of FITC and EDC do not overlap. Consequently, there is no indication of dual positive cells.
  • FIG. 2 results were obtained is the same manner as realized for FIG. 1, except that there was compensation for overlapping wavelengths.
  • FIG. 2A illustrates the gating of the preserved cells on the basis of forward light scatter (FS) and log side light scatter (LSS) included in electronic gate (1) .
  • FS forward light scatter
  • LSS log side light scatter
  • FIG. 2B illustrates the correct fluorescent reading which is obtained after setting the appropriate compensation for overlapping fluorescent emissions using antigenically preserved cells as control cells.
  • FIG. 2C illustrates that when there is appropriate fluorescence compensation, no LFL3 positive cells are indicated in Quads 1 and 2.
  • FIG. 2D (LFL3/LFL1) illustrates that with appropriate fluorescence compensation, no LFL3 positive cells are indicated in Quads 1 and 2.
  • preserved cells can be used to adjust the instrument's light scatter to incorporate the preserved cell population of interest, for example, lymphocytes and sub-populations thereof.
  • the mean channel of the positive population and the mean channel of the negative population should be equal or nearly equal. This means that for the phycoerythrin-RDl fluorescence, the mean channel of the fluorescence of the cells labelled with FITC should be equivalent or nearly equivalent to the mean channel of the negative cells labelled with phycoerythrin RD1.
  • the mean channel of the fluorescence of the cells labelled with phycoerythrin-RDl should be equivalent or nearly equivalent to the mean channel of the negative cells labelled with FITC.
  • the procedure can be used with two or more fluorescent labels.
  • the use of antigenically preserved cells, as shown in the Examples, enables the compensation factor to be reproducible over time and between different instruments and different laboratories.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention describes a method of using differently labelled, reconstituted preserved cells as control cells in multiple color assays. These cells are used to calibrate light scatter and fluorescent intensity measurements. The use of said control cells overcomes the preservation and calibration difficulties experienced in the conventional use of fresh cells and polymer beads as controls in determining the compensation adjustments required in multiple color flow cytometry assays as a result of the overlapping wavelength regions encountered.

Description

METHOD OF USING PRESERVED CONTROL CELLS IN THE CALIBRATION OF FLUORESCENT AND LIGHT SCATTER MEASUREMENTS
TECHNICAL FIELD The invention relates to the use of labelled preserved cells as controls to make compensation adjustments for the overlapping wavelength emissions of multiple fluorescent substances used as labels in multiple color biological analyses by flow cytometry. The invention also describes the preparation of preserved labelled cells which have stability and shelf life characteristics not previously realized. BACKGROUND ART
Control cells are essential for the accuracy and precision of clinical tests and immunoassays. In assays which use light scatter and fluorescent labels to identify cells and/or cellular components, control cells are used to form the basis for making compensation adjustments for areas of fluorescent overlap. The use of control cells and the making of the compensation adjustments is necessary to insure the reliability and accuracy of the test equipment and methods, and to insure reproducibility through time and from 1aboratory-to- laboratory.
In assays using light scatter and fluorescent labels, cells and/or cellular substances are determined on the basis of either direct interaction with the fluorescent substance or indirect interaction via bonding of the fluorescent substance to a biological substance, for example, a monoclonal antibody, plant lectin and other substances which may be involved in the reaction with the cells or cellular components. When only a single fluorescent substance is used in an assay, any • compensation adjustments are not difficult to do. However, such adjustments become very important and are more difficult when multiple fluorescent labels are utilized in multiple color analysis. The problem arises because many of the labels used in these analyses have overlapping emission wavelength regions. The fluorescent light produced in the overlap region is the sum of the light produced by overlapping wavelengths of the individual fluorescent substances. The problem is particularly important in flow cyto etry where the objective is to label cells with at least two distinct fluorochro es which reflect discrete cellular components by detecting a signal proportional to each fluorochrome and unbiased by any contribution from the other fluorochrome. Unless adjustments are made for the overlapping wavelengths, false "positive" readings may occur. It is not possible to choose wavelength filters which will optimally transmit the light of one overlapping] fluorochrome and totally block the light of the other fluorochrome, thereby avoiding the false positive readings. Most multiple color analytical systems provide "compensation" to eliminate the false positive readings. Compensation, as used herein, is an electronic means of computing and subtracting overlapping fluorescent signals, i.e., the overlap contribution from a second fluorochrome is subtracted from the fluorescence reading of a first fluorescent label on the cells being analyzed. When cells bearing the second fluorochrome are analyzed, the process is reversed and the overlap contribution from the first fluorochrome is subtracted from the fluorescence reading of the second fluorescent label. In order to determine the proper amount of electronic compensation, there must be some method of determining whether too much or too little signal is subtracted relative to the specific fluorochrome and the targeted cells or cellular components.
Traditionally, fluorescently labelled fresh normal blood samples or latex beads have been used as controls in light scatter analyses to determine the necessary adjustments and the appropriate amount of electronic compensation. However, fresh normal blood samples are not entirely satisfactory because they can be used as a compensation controls for only a short period of time due to the degradation which such samples undergo during storage. New normal blood samples must be obtained continuously. As a result, fresh blood samples are neither amenable to commercialization because of their lack of stability nor do they provide a uniform baseline for the comparison of samples separated in time. Latex beads offer better stability and a uniform baseline, but their light scatter characteristics are different from the light scatter characteristics of biological cells or cellular components. Consequently, the use of latex beads provides compensation values which are different from those values obtained using real cells. Another consideration in flow cytometry is that is important that the size, shape and structure of the labelled calibration substance have a direct relationship to the size, shape and structure of the cells or cellular components being analyzed. For example, a large labelled spherical calibration or control particle would not be a proper match for an unknown consisting of small rectangular particles. .For this reason, biological cells are preferred over latex beads as controls.
This invention provides a method for using preserved, labelled biological cells as control cells for light scatter measurements and fluorescent calibration. A provision providing for such cells enables the analyst to select the proper cells for making compensation adjustments and provides the ability to establish long term baselines for the comparison of samples analyzed at different points in time.
DISCLOSURE OF INVENTION The invention describes a method of using labelled reconstituted preserved cells as control cells to calibrate light scatter and fluorescent intensity measurements in multiple color assays using two or more fluorescent labels and to provide for compensation adjustments in such assays which eliminate false positive readings caused by the overlapping wavelengths of different fluorescent substances. The compensation factors used in the adjustments are determined electron¬ ically by the analytical instrument. The compensation factors prevent the appearance of false positive readings during the assay.
The assays with which the method is used are biological cells assays. The method is an improvement over conventional techniques which require fresh cells or which use polymeric beads. The use of labelled preserved cells according to the invention enables the analyst to establish a long term baseline for the comparison of samples assayed at different points in time. The invention also describes preferred labelled preserved cells for use as the control cells and a method of preparing the same.
BRIEF DESCRIPTION OF DRAWINGS FIG 1A illustrates the gating, with no compensation adjustments, of the preserved cells on the basis of forward light scatter (FS) and log side light scatter. FIG. IB illustrates false positive fluorescent staining of cells in Quad 2 due to lack of fluorescent compensation adjustments.
FIG. 1C further illustrates false positive fluorescent staining in Quad 2 due to lack of compensation adjustments.
FIG. ID illustrates that even without compensation adjustments there is no overlap region for FITC and ECD fluorescent emissions. FIG. 2A illustrates the gating, with compensation adjustments of the preserved cells on the basis of forward light scatter and log side light scatter.
FIG. 2B illustrates the correct fluorescent reading obtained after sitting the appropriate compensation for overlapping fluorescent emissions.
FIG. 2C illustrates that with the appropriate fluorescent compensation for overlapping emission wavelengths, there are no false positive readings in Quads 1 and 2.
FIG. 2D further demonstrates that with the appropriate fluorescent compensation, these are no false positive readings in Quads 1 and 2. BEST MODE FOR CARRYING OUT THE INVENTION
The invention describes a method which uses pre¬ served cells to determine light scatter and compensation adjustments for wavelength emissions of multiple fluorescent substances used as labels in multiple color biological analysis. In general, any preserved, labelled cells which retain their authentic structural and antigenicity characteristics can be used according to the invention. For example, one may practice the invention by using lyophilized labelled cells as control cells. Lyophilized cells may be prepared according to U.S. Patent No. 5,059,518 issued October 29, 1991 for STABILIZED LYOPHILIZED MAMMALIAN CELL AND METHOD OF MAKING SAME. The teachings of Patent No. 5,059,518 are incorporated herein by reference. Other methods of preserving cells, bacteria, and other biological substance may be found in U.S. Patent Nos. 3,261,761, 4,874,690, 4,206,200 and 4,246,349; European Patent Application No. 0 259 736, published March 16, 1988; and International Patent Applications published under the Patent Cooperation Treaty as Nos. WO 86/03938 (published July 17, 1986), WO 87/00196 (published January 17, 1987) and WO 89/06976 (published August 10, 1990); and Japanese Patent Application Nos. 57-7419, 56-12317 and 58-131913. The selection criteria for any preserved cells used according to the invention is that when such cells are reconstituted, washed or other prepared for use, they retain their authentic structural and antigenic characteristics, and that these characteristics are the same as corresponding fresh cells. Such preserved cells, after reconstitution, washing or otherwise being prepared for use in the claimed invention are hereby defined as "reconstituted control cells" or simply "control cells." The labels used in the invention are fluorescent substances. The labelling may be done by direct reaction between the label and biological cells or cellular components. Alternatively, the label may be attached to (1) a reactive biological material, such as, a monoclonal antibody or (2) a reactive non-biological organic substance, for example, a carboxylic acid chloride, prior to attachment to the cells or cellular components. For example, fluorescent labels may be attached to a selective reactant substance such as a monoclonal antibodies and the labelled monoclonal antibodies then conjugated to cells. The preferred method is to attach the label to a selectively reactive substance, and most preferably, to a monoclonal antibody.
The fluorescent labels may be selected from fluoro- scein, fluoroscein isothiocyanate (FITC), rhodamine, tetramethylenerhodamine isothiocyanate (TRITC), sulfor- hodamine 101 acid chloride (Texas Red), phycoerythrin (PE), allophycocyanin, phycoerythrin-Texas Red (PETR), 4-methylumbelliferone and other fluorescent substances known or found useful in the analysis of biological substances. The conjugation of the label to the monoclonal antibody can be performed by any suitable and known method including direct reaction between the monoclonal antibody and the label or the use of bridging groups to connect the monoclonal antibody and the label. The labelled lyophilized cells used herein as control cells may be prepared by reconstituting cells lyophilized according to Patent No. 5,059,518 and incubating the reconstituted cells with either a polyclonal or monoclonal antibody. Monoclonal antibodies specific to an antigenic site present on the cells are preferred. The antibody may be labelled before incubation with the reconstituted cells or it may be labelled after it has been attached to the reconstituted cells. The former is preferred. The resulting labelled cells are then used according to the claimed invention.
Alternatively, fresh cells may be labelled, for example, by incubating the fresh cells with an antibody. The antibody may be labelled before or after incubation. Again, the former is preferred. After labelling, the cells are lyophilized according to said Patent No. 5,059,518. It has been determined that preservation by the method of said patent does not destroy or decrease the fluorochrome's fluorescent ability as is known to occur using other methods of preservation. Prior to their use as control cells according to the herein invention, the lyophilized labelled cells are reconstituted.
It is also within the scope of the invention to use cells which have been labelled directly. Directly labelled cells are those in which the label is attached to the cell without the use of an intervening antibody or other substance.
Example Of Practicing The Invention. An anti-CD8 monoclonal was labelled with phycoerythrin (RD1, obtainable from Coulter Corporation of Miami, Florida) to produce a labelled antibody designated as LFL2. An anti-CD4 monoclonal antibody was labelled with fluoroscein isothiocyanate (FITC) to produce a species designated as LFL1. Lastly, a negative control antibody (IgG b) was labelled with ECD (energy coupled dye, Coulter Corporation, Miami, Florida) to produce a species designated LFL3. (All antibodies are available from Coulter Corporation, Miami, Florida). These three labelled antibody species were used to label lymphocytes preserved by the method described in patent No. 5,059,518. Tricolor (RD1, FITC, EDC) flow cytometric analysis of the cells using this plurality of labels was then performed using no compensation as illustrated in Fig. 1 and using compensation as illustrated in Fig. 2. As used herein the use of a "plurality" of fluorescent labels signifies the use of two or more labels. The maximum number is dependent solely on the capabilities of the instrument used in the assay, i.e. it may be two, three, four or more labels. FIG. 1A illustrates the gating of the preserved cells on the basis of forward light scatter (FS) and log side light scatter (LSS) included in electronic gate (1). FIG. IB (LFL2/LFL1) illustrates the false positive fluorescent staining of cells in Quadrant (Quad) 2 due to the lack of fluorescence compensation, such that, there appears CD8-FITC positive cells in Quadrant 1 (LFL2+); CD4-RD1 positive cells in Quadrant 4 (LFL1+); LFLl(-) LFL2(-) cells in Quadrant 3; and LFL1(+) LFL2(+) cells in Quadrant 2. The dual positive cells seen in Quadrant 2 are the result of the FITC emission wavelength overlapping into LFL2 (overlap between FITC and RD1 emissions) . It should be noted that the mean channel of LFL2 in Quad 3 (x-axis = 0.146) is quite different from LFL2 in Quad 1 (x-axis = 0.233) and that LFL1 in Quad 3 (y-axis = 0.128) is quite different from LFL1 in Quad 4 (y-axis = 0.222).
FIG. 1C (LFL3/LFL2) illustrates the false positive fluorescence is due to the fact that there is no fluorescence compensation for overlapping wavelengths (refer to FIG. IB). The dual positive cells seen in Quadrant 2 are the result of the emission wavelength from LFL2 (RD1 emission) overlapping with LFL3 (ECD emission). FIG. ID (LFL3/LFL1) illustrates that without compensation, the separate emissions of FITC and EDC do not overlap. Consequently, there is no indication of dual positive cells.
FIG. 2 results were obtained is the same manner as realized for FIG. 1, except that there was compensation for overlapping wavelengths. FIG. 2A illustrates the gating of the preserved cells on the basis of forward light scatter (FS) and log side light scatter (LSS) included in electronic gate (1) .
FIG. 2B (LFL2/LFL1) illustrates the correct fluorescent reading which is obtained after setting the appropriate compensation for overlapping fluorescent emissions using antigenically preserved cells as control cells. It should be noted that the mean channel of LLF2 in Quad 3 (x-axis = 0.169) LFL2 in Quad 1 (x-axis = 0.150) are similar. Likewise, the mean channel of LFL1 in Quad 3 (y-axis = 0.147) is similar to the mean channel of LFL1 in Quad 4 (y-axis = 0.143). In contrast to FIG. IB, there are no false positive readings.
FIG. 2C (LFL3/LFL2) illustrates that when there is appropriate fluorescence compensation, no LFL3 positive cells are indicated in Quads 1 and 2.
FIG. 2D (LFL3/LFL1) illustrates that with appropriate fluorescence compensation, no LFL3 positive cells are indicated in Quads 1 and 2.
As seen in FIGS. 2A-2D, preserved cells can be used to adjust the instrument's light scatter to incorporate the preserved cell population of interest, for example, lymphocytes and sub-populations thereof. To verify that the color compensation is appropriate for a given signal, the mean channel of the positive population and the mean channel of the negative population should be equal or nearly equal. This means that for the phycoerythrin-RDl fluorescence, the mean channel of the fluorescence of the cells labelled with FITC should be equivalent or nearly equivalent to the mean channel of the negative cells labelled with phycoerythrin RD1. For the FITC fluorescence, the mean channel of the fluorescence of the cells labelled with phycoerythrin-RDl should be equivalent or nearly equivalent to the mean channel of the negative cells labelled with FITC. When the analytical instrument is so adjusted, the correct overlapping wavelength compensation is achieved and used in the analysis.
The procedure can be used with two or more fluorescent labels. The use of antigenically preserved cells, as shown in the Examples, enables the compensation factor to be reproducible over time and between different instruments and different laboratories. We claim:

Claims

AMENDED CLAIMS
[received by the International Bureau on 25 May 1994 (25.05.94); original claims 1,2,5,7 and 11 amended; remaining claims unchanged (3 pages)]
1. A method of calibrating a flow cytometer instrument for use in a predetermined multiple color assay of a sample containing at least two types of cells, each type of which has been labelled with a different
5 fluorescent dye, said different dyes having overlapping excitation wavelength regions, said method comprising:
(a) preparing as control cells at least two types of biological cells selected to be suitable for conducting said multiple color assay, each of said type
10 of cells having a different fluorescent label conjugated thereto, said labelled control cells having different but overlapping excitation wavelength regions and comprising reconstituted preserved cells having the desired biological characteristics for said assay and for
15 measuring said overlapping wavelength regions when said cells are processed through a flow cytometer;
(b) processing said labelled control cells through said flow cytometer to ascertain relevant measurements which denote the overlapping excitation wavelength
20 region;
(c) using said relevant measurements to determine the required compensation adjustments to be used in conducting the assay of the selected biological cells with said flow cytometer; and
25 (d) making said compensation adjustments.
2. A method of calibrating a flow cytometer for use in a predetermined multiple color assay of a sample containing at least two different types of biological cells which have been labelled with fluorescent dyes that
30 have different, but partially overlapping emission wavelength regions, said method comprising:
(a) preparing test samples containing at least two types of control cells selected as suitable for conducting said multiple color assay, and within a given
35 test sample only one type of cells has only one of said fluorescent dyes conjugated thereto, said control cells consisting of reconstituted preserved cells having the required biological characteristics necessary for performing said predetermined assay and labelled with said fluorescent dye either before preservation or after reconstitution;
(b) processing said labelled control cell samples sequentially through said cytometer to determine the relevant intensity measurements for each cell-conjugated dye throughout its emission spectrum; and (c) making the necessary compensation adjustments to said cytometer using the measurements of step (b) in order to obtain an accurate assay within the overlapping region of the emission spectra of said dyes; wherein when said method is completed, said flow cytometer is calibrated to accurately assay cells within a sample in which the same type of cells are labelled with said fluorescent dyes.
3. The method of claim 2 wherein said control cells are selected from the group consisting of: (a) cells in which the dyes are attached directly to said cells and
(b) cells in which the dyes are attached to said cells by means of a bridging group between said dye and said cells. 4. The method of claim 3 wherein said bridging group is an antibody.
5. The method of claim 3 wherein a single type of cells is classified into subsets by labelling said type of cells with at least two selected fluorescently labelled monoclonal antibodies, each of said fluorescent labels being on a different antibody.
6. The method of claim 3 wherein said control cells are labelled, reconstituted lyophilized cells.
7. The method of claim 1 in which at least an additional dye-labelled control cell is prepared for use in the assay which does not overlap in the spectrum of the two dye-labelled control cells at the excitation wave length used to excite said two dye-labelled control cells.
8. The method of claim 1 in which said control cells are labelled, reconstituted lyophilized cells.
9. The method of claim 8 wherein said control cells are selected from the group consisting of:
(a) cells in which the dyes are attached directly to said cells and
(b) cells in which the dyes are attached to said cells by means of a bridging group between said dye and said cells.
10. The method of claim 9 wherein said bridging group comprises an antibody.
11. The method of claim 1 in which at least an additional dye-labelled control cell prepared for use in the assay which does overlap in the spectrum of only one of the two of dye-labelled control cells at the excita¬ tion wavelength used to excite said two dye-labelled control cells.
EP94906567A 1993-01-08 1994-01-07 Method of using preserved control cells in the calibration of fluorescent and light scatter measurements. Withdrawn EP0678195A4 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US183493A 1993-01-08 1993-01-08
US1834 1993-01-08
PCT/US1994/000295 WO1994016314A1 (en) 1993-01-08 1994-01-07 Method of using preserved control cells in the calibration of fluorescent and light scatter measurements

Publications (2)

Publication Number Publication Date
EP0678195A1 EP0678195A1 (en) 1995-10-25
EP0678195A4 true EP0678195A4 (en) 1998-07-08

Family

ID=21698048

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94906567A Withdrawn EP0678195A4 (en) 1993-01-08 1994-01-07 Method of using preserved control cells in the calibration of fluorescent and light scatter measurements.

Country Status (5)

Country Link
EP (1) EP0678195A4 (en)
JP (1) JPH08510051A (en)
AU (1) AU6024294A (en)
CA (1) CA2153344A1 (en)
WO (1) WO1994016314A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010018192A1 (en) 1998-02-12 2001-08-30 Terstappen Leon W.M.M. Labeled cells for use as an internal functional control in rare cell detection assays
FR2895087B1 (en) * 2005-12-20 2008-02-08 Horiba Abx Sas Soc Par Actions METHOD OF DISCRIMINATION OF AT LEAST TWO CELLULAR POPULATIONS AND APPLICATION
US11137388B2 (en) 2014-01-14 2021-10-05 Asedasciences Ag Identification of functional cell states

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4987086A (en) * 1987-11-30 1991-01-22 Becton, Dickinson And Company Method for analysis of subpopulations of cells
US5270548A (en) * 1992-07-31 1993-12-14 The United States Of America As Represented By The United States Department Of Energy Phase-sensitive flow cytometer

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5059518A (en) * 1988-10-20 1991-10-22 Coulter Corporation Stabilized lyophilized mammalian cells and method of making same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4987086A (en) * 1987-11-30 1991-01-22 Becton, Dickinson And Company Method for analysis of subpopulations of cells
US5270548A (en) * 1992-07-31 1993-12-14 The United States Of America As Represented By The United States Department Of Energy Phase-sensitive flow cytometer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9416314A1 *

Also Published As

Publication number Publication date
JPH08510051A (en) 1996-10-22
CA2153344A1 (en) 1994-07-21
WO1994016314A1 (en) 1994-07-21
AU6024294A (en) 1994-08-15
EP0678195A1 (en) 1995-10-25

Similar Documents

Publication Publication Date Title
Kettman et al. Classification and properties of 64 multiplexed microsphere sets
CA1248856A (en) Method and reagent system for four-population differential determination of leukocytes
EP2187199B1 (en) Instrument setup method for a fluorescence analyzer
US5084394A (en) Method for corrective calibration of a flow cytometry using a mixture of fluorescent microbeads and cells
EP0469766B1 (en) Preservation of cells as controls or standards in cellular analysis
EP0258983A2 (en) Method for calibrating flow cytometers
JPH04252953A (en) Data converting method
WO1991000509A1 (en) Method and microbeads for calibrating a flow cytometer
Franck et al. Measurement of intracellular pH in cultured cells by flow cytometry with BCECF-AM
Tannenbaum et al. Measurement of cellular DNA mass by flow microfluorometry with use of a biological internal standard.
JPH0464429B2 (en)
Bohn Flow cytometry: a novel approach for the quantitative analysis of receptor-ligand interactions on surfaces of living cells
CN101126758A (en) Flow cytometry synchronous detection method for multiple protein expression of tumor cell
Purvis et al. Multi‐platform, multi‐site instrumentation and reagent standardization
US4714672A (en) Immunoassays involving complement lysing of chromophore containing microcapsules
CA1279008C (en) Methods and reagents for performing subset analysis
EP0678195A4 (en) Method of using preserved control cells in the calibration of fluorescent and light scatter measurements.
Zhang et al. Novel flow cytometry compensation standards: Internally stained fluorescent microspheres with matched emission spectra and long‐term stability
US20040214243A1 (en) Differential determination of hemoglobins
CN110133280A (en) A kind of measuring method of the glycation ratio of hemoglobin of β chain variation
CN108693099A (en) Full-automatic fluorescence compensation method for flow cytometer
CN111528219B (en) Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof
US20110189777A1 (en) Methods and compositions for improved diagnostic assays
US5968831A (en) Cell control used to confirm enzymatic activity
Cram et al. A quantitative method for evaluating fluorescent antibodies and the conjugation process

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19950721

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): DE ES FR GB

RIN1 Information on inventor provided before grant (corrected)

Inventor name: JOHNSON, MARCIA

Inventor name: GUPTA, RAVINDER, K.

Inventor name: MAPLES, JOHN, A.

RHK1 Main classification (correction)

Ipc: G01N 33/50

A4 Supplementary search report drawn up and despatched

Effective date: 19980522

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): DE ES FR GB

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 19980720