EP0678195A1 - Procede d'utilisation de cellules temoins dans l'etalonnage de mesures de l'intensite fluorescente et de la dispersion de lumiere - Google Patents

Procede d'utilisation de cellules temoins dans l'etalonnage de mesures de l'intensite fluorescente et de la dispersion de lumiere

Info

Publication number
EP0678195A1
EP0678195A1 EP94906567A EP94906567A EP0678195A1 EP 0678195 A1 EP0678195 A1 EP 0678195A1 EP 94906567 A EP94906567 A EP 94906567A EP 94906567 A EP94906567 A EP 94906567A EP 0678195 A1 EP0678195 A1 EP 0678195A1
Authority
EP
European Patent Office
Prior art keywords
cells
labelled
control cells
assay
dye
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Withdrawn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)
English (en)
Other versions
EP0678195A4 (fr
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/fr
Publication of EP0678195A4 publication Critical patent/EP0678195A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1012—Calibrating particle analysers; References therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical 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.

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  • 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

L'invention concerne un procédé d'utilisation de cellules préservés reconstituées marquées différemment en tant que cellules témoins dans des méthodes de coloration multiple. Ces cellules sont utilisés pour étalonner des mesures de dispersion de la lumière et d'intensité fluorescente. L'utilisation desdites cellules témoins supprime les difficultés de conservation et d'étalonnage rencontrées lorsque des cellules fraîches et des perles de polymère sont utilisées en tant que témoins pour déterminer les réglages de compensation nécessaires dans les méthodes de cytométrie de flux à coloration multiples en fonction des régions de longueur d'ondes se chevauchant.
EP94906567A 1993-01-08 1994-01-07 Procede d'utilisation de cellules temoins dans l'etalonnage de mesures de l'intensite fluorescente et de la dispersion de lumiere. Withdrawn EP0678195A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US1834 1987-01-09
US183493A 1993-01-08 1993-01-08
PCT/US1994/000295 WO1994016314A1 (fr) 1993-01-08 1994-01-07 Procede d'utilisation de cellules temoins dans l'etalonnage de mesures de l'intensite fluorescente et de la dispersion de lumiere

Publications (2)

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

Family

ID=21698048

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94906567A Withdrawn EP0678195A4 (fr) 1993-01-08 1994-01-07 Procede d'utilisation de cellules temoins dans l'etalonnage de mesures de l'intensite fluorescente et de la dispersion de lumiere.

Country Status (5)

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

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 (fr) * 2005-12-20 2008-02-08 Horiba Abx Sas Soc Par Actions Methode de discrimination d'au moins deux populations cellulaires et application
KR102413041B1 (ko) 2014-01-14 2022-06-23 아세다사이언시즈 아게 기능성 세포 상태의 확인

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 (ja) 1996-10-22
EP0678195A4 (fr) 1998-07-08
AU6024294A (en) 1994-08-15
CA2153344A1 (fr) 1994-07-21
WO1994016314A1 (fr) 1994-07-21

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