EP0745217A1 - Sondes de viabilite pour isoler, identifier et/ou analyser des cellules - Google Patents
Sondes de viabilite pour isoler, identifier et/ou analyser des cellulesInfo
- Publication number
- EP0745217A1 EP0745217A1 EP95907435A EP95907435A EP0745217A1 EP 0745217 A1 EP0745217 A1 EP 0745217A1 EP 95907435 A EP95907435 A EP 95907435A EP 95907435 A EP95907435 A EP 95907435A EP 0745217 A1 EP0745217 A1 EP 0745217A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- further characterized
- probe
- viability
- staining
- 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- Withdrawn
Links
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
Definitions
- the present invention provides viability probes which can be use independently or are useful for inclusion in a monoclonal antibody panel for the analysis of solid or non-adherent tumors.
- Non-viable, permeabilized cells constitute a significant source of interference in flow cytometric analysis. This interference results primarily from differential antigen expression (13) , biochemical changes (e.g., increased autofluorescence) (24), and the nonspecific uptake and binding of probes due to membrane permeabilization accompanying cell death (6,15,20,22).
- necrosis and apoptosis are necrosis and apoptosis (1,3,5,9,13,19).
- Necrosis is typically induced by extracellular conditions, such as hypoxia or cytotoxic agents, and is characterized by a sequence of metabolic collapse, cell swelling, lysis, and inflammation of surrounding tissue (1,5,7,9,23).
- the more common phenomenon of apoptosis (1,10) is associated with programmed cell death, and is characterized by a sequence of cell shrinkage, chromatin condensation and cleavage, and finally, membrane permeabilization (1,3,5,7,9,10,13).
- As dying cells permeabilize changes in their morphology and refractive index significantly alter the cells' light scatter characteristics, and the loss of membrane integrity permits uptake of normally impermeant probes.
- flow cytometric dead cell discrimination techniques can be divided into three main categories: (1) physical separation methods, (2) differential light scatter, and (3) differential staining by fluorescent probes. These techniques are largely measures of membrane permeabilization, and are based on the equation of this phenomenon with cell death (6,13).
- permeabilizing agents as is often necessary for sample preservation, intracellular labelling, or biohazard control, presents a significant challenge to the discrimination of naturally permeabilized dead cells from cells that were alive prior to artificial permeabilization.
- Such agents can drastically alter cellular morphology and refractive index, resulting in homogenization of the light scatter characteristics of live and dead cells, as well as cells of different types.
- permeabilizing agents allow all cells, including those that were live prior to permeabilization, to take up any probe present in excess in the solution, or that leaks back out of previously stained dead cells.
- fixation and permeabilization generally preclude the use of the two most common fresh prep techniques for dead cell discrimination, i.e., electronic gating on the basis of light scatter and uptake of membrane-impermeant probes.
- physical separation methods such as percoll and ficoll density gradient separation can separate dead from live cells on the basis of differing buoyant densities (5) , but have the disadvantages of wide variations in purity and recovery, selective cell loss (11) , and in some circumstances, cell toxicity (21) .
- the present invention provides viability probes for inclusion in solid tumor monoclonal antibody panels, which established the following criteria: broad cell type specificity; low nonspecific staining of live cells; high signal-to-noise staining of dead cells; binding in dead cells of sufficiently high affinity to withstand the permeabilization procedure; and excitation and emission spectra compatibility with the other dyes in the solid tumor panel.
- the viability probes of the present invention may be use in the methods of application having docket number
- FIG. 1 shows a sample processing flow chart
- FIG. 2 shows a comparison of light scatter (LT SCATTER) and PI staining of unprocessed cells with staining of processed cells by experimental viability probes actin-SAM-FITC (ACTIN-SF) , cytokeratin-SAM-FITC (CK-SF) , 7-AAD, and TO-PRO-3.
- actin-SAM-FITC ACTIN-SF
- CK-SF cytokeratin-SAM-FITC
- 7-AAD 7-AAD
- TO-PRO-3 TO-PRO-3
- FIG.3 shows a comparison of light scatter (LT SCATTER) and PI staining of unprocessed cells (PI UNFIXED) with staining of processed cells by experimental viability probes LDS-751 (LDS) , tubulin-SAM-FITC (TUBULIN-SF) , EMA, and PI (PI FIX) .
- LDS-751 LDS-751
- TUBULIN-SF tubulin-SAM-FITC
- EMA PI
- PI FIX PI FIX
- the present invention provides viability probes which are useful for inclusion in a monoclonal antibody panel for the analysis of solid, preferably breast, tumors.
- the viability probes of the present invention demonstrate sufficiently broad cell type specificity to be informative for the large variety of cells found in solid tumors (4) ; low nonspecific binding; specific binding strong enough to withstand the staining and permeabilization procedures; both high and stable fluorescence intensity, to remain informative over the period of time dead cells may persist in a solid tumor before disintegration (at which point the debris can be discriminated or gated out on the basis of light scatter) ; and compatibility with the four other fluorescent probes comprising the tumor antibody panel.
- tubulin-SAM-FITC appears to satisfy all of the aforementioned criteria: the presence of tubulin in all eukaryotic cells (18) circumvents the problem of tumor heterogeneity; the probe exhibits little or no nonspecific binding to intact cells, and its binding in permeabilized cells is sufficiently strong to withstand the staining and permeabilization procedures; its signal-to-noise ratio is high enough to clearly distinguish it from autofluorescence even through day 15 after cell harvest, and its fluorescence intensity diminishes relatively slowly over the time course; and it is compatible with the other probes comprising the tumor antibody panel. Cytokeratin-SAM-FITC was satisfactory in every respect other than its specificity only for epithelial cells.
- the present invention supports this categorization.
- Types I and III dead cells is suggested by the fact that staining by DNA-specific probes is most intense at the onset of cell death and decreases with increasing cell age after harvest.
- Type II dead cells, with intact membranes and degraded chromatin is predicted by the consistently higher percentages (Tables 2 and 3) of cells with low forward scatter (suggesting the loss of cell volume that precedes chromatin degradation in apoptosis) (7) than positivity for the viability probes (excluding TO-PRO-3, LDS-751, and PI in processed cells, all of which stained live and dead cells indiscriminately) .
- the viability probes of the present invention are further directed against a relatively stable intracellular antigen which proves more informative than traditional, DNA-specific viability dyes (e.g., propidium iodide) for late apoptotic and necrotic cells containing degraded chromatin (Type III) , as well as permeabilized cells containing native chromatin (Type I) .
- a relatively stable intracellular antigen which proves more informative than traditional, DNA-specific viability dyes (e.g., propidium iodide) for late apoptotic and necrotic cells containing degraded chromatin (Type III) , as well as permeabilized cells containing native chromatin (Type I) .
- the present invention has the significant advantage of being resistant to fixation and permeabilization procedures, such as are often required for sample preservation, biohazard control, or intracellular labelling.
- the present invention teaches the use of an antibody probe, which can be conjugated to any of a wide array of available fluorophores
- the viability probe is cytokeratin. In the most preferred embodiment of the present invention the viability probe is tubilin.
- the present invention provides two time-course studies presented below, wherein tumor cell lines were induced to enter a condition of cellular hypoxia intended to simulate the internally hypoxic environment of a solid tumor, and in which their degree of staining by numerous viability probes is compared.
- the first study involved staining of the MDA (breast tumor) cell line by propidium iodide (PI) without permeabilization as the standard, and, with permeabilization, anti-actin-SAM-FITC, anti- cytokeratin-SAM-FITC, 7-aminoactinomycin D (7-AAD; a high- affinity DNA intercalator with an emission maximum at approximately 650 nm) (16,17), and T0-PR0-3 (a membrane- impermeant, high-affinity DNA intercalator with an emission maximum at 661 nm, and minimal fluorescence except when bound to nucleic acids) .
- PI propidium iodide
- the second study included staining of MDA cells by PI without permeabilization as the standard, and, with permeabilization, anti-tubulin-SAM-FITC, ethidiu monoazide bromide (EMA, a photoactivated, covalently binding DNA intercalator with an emission maximum at approximately 600 nm) (15) , LDS-751 (a vital nucleic acid stain with an emission maximum at 670 nm) (20), and PI.
- EMA ethidiu monoazide bromide
- PBS lysophosphatidyl choline
- Nonidet P-40 NP40
- trypan blue anti- ⁇ -smooth muscle actin monoclonal antibody
- 7-aminoactinomycin D 7-AAD
- Propidium iodide (PI) stain (from the DNA-Prep Kit; contains 50 ug/ml PI, 4 KU/ml bovine pancreas Type III RNAse, 0.1% NaN 3 , saline, and stabilizers) and anti-cytokeratin monoclonal antibody were provided by Coulter Corporation (Miami, Florida) .
- Anti- ⁇ -tubulin was purchased from Zymed Laboratories, Inc. (San Francisco, CA) .
- FITC-conjugated sheep-anti-mouse F(ab') fragments (SAM-FITC) were obtained from Silenus Laboratories (Victoria, Australia) .
- Ethidium monoazide bromide (EMA) and T0-PR0-3 were from Molecular Probes, Inc. (Eugene, OR) .
- LDS-751 was purchased from Exciton (Dayton, OH) .
- samples were processed according to the procedure used in our laboratory for simultaneous surface and intranuclear staining of solid tumors.
- the unprocessed series to be stained with PI and their unstained controls were centrifuged at 500 x g for 5 minutes at room temperature and decanted.
- the unstained controls were resuspended in PBS supplemented with 2.5% FBS (PBSF) , and the Pi-stained samples were resuspended in 1 ml and incubated in darkness at room temperature for twenty minutes prior to flow cytometric analysis.
- PBSF 2.5% FBS
- 7-AAD-stained cells were resuspended in 1 ml/test of 25 ⁇ g/ml 7-AAD (250 ⁇ l of stock solution in 49.75 ml PBSF; stock solution is 5 mg/ml in methanol) and incubated in darkness for 15 minutes at room temperature.
- TO-PRO-3- stained cells were resuspended in 1 ml/test of 5 ⁇ M TO- PRO-3 in PBSF and incubated in darkness for 15 minutes at room temperature.
- LDS-751-stained cells were resuspended in 1 ml/test of PBSF containing 10 ⁇ l of 2 ⁇ g/ml LDS-751 (50 ⁇ l of stock solution in 4.95 ml PBSF; stock solution is 0.2 mg LDS-751/ml methanol) and incubated in darkness for 15 minutes at room temperature.
- EMA-stained cells were resuspended in 10 ⁇ l/test of 5 ⁇ g/ml EMA (125 ⁇ l of stock solution in 4.875 ml PBSF; stock solution is 0.2 mg EMA/ml methanol) and incubated for 15 minutes at room temperature, at a distance of 20 cm from a fluorescent light bulb.
- Pi-stained cells were resuspended in 1 ml/test of the same PI solution used for unprocessed cells and incubated in darkness for 15 minutes at room temperature. All samples were then washed as above, followed by resuspension in 200 ⁇ l/test PBSF, and a 15- minute, room-temperature incubation in darkness (to simulate surface antigen staining of tumor samples) . All samples were then washed twice more as above.
- each sample was resuspended in 1 ml of 25°C 20 ⁇ g/ml lysophosphatidyl choline in 1% paraformaldehyde, incubated for 2 minutes at room temperature, centrifuged at 500 x g for 5 minutes at room temperature, and decanted. The samples were then resuspended in 1 ml/test of -20°C absolute methanol and incubated on ice for 10 minutes, followed by centrifugation as above. After decanting, the samples were resuspended in 1 ml/test of 4°C 0.1% NP40 and incubated on ice for 5 minutes, followed by centrifugation as above.
- samples were resuspended in 200 ⁇ l/test of PBSF and incubated in darkness for 15 minutes at room temperature (to simulate intracellular antigen staining of tumor samples) , followed by washing as above. Finally, the samples were resuspended in 1 ml/test PBSF prior to flow cytometric analysis.
- Fluorescence emission of actin-, cytokeratin-, and tubulin-SAM-FITC was reflected by a 550 nm dichroic long- pass filter and passed through a 525 nm bandpass filter.
- PI and EMA fluorescence were reflected by a 650 nm dichroic long-pass filter and passed through a 610 nm bandpass filter.
- 7-AAD, TO-PRO-3, and LDS-751 fluorescence passed through 650 nm dichroic long-pass and 675 nm bandpass filters.
- Autofluorescence of unstained controls was measured through the same filter configurations as the stained samples with which they were matched.
- the next column displays light scatter results for unprocessed cells from each batch (in each of these histograms, forward scatter is on the Y axis, and side scatter is on the X axis) .
- the remaining columns display fluorescence histograms of unstained cells (in green) electronically overlaid with histograms (in colors other than green) of cells stained with the probe indicated at the top of each column. All instrument settings remained unchanged for the duration of the experiment. Fluorescence histograms are on a four-decade logarithmic scale. All histograms contain 5,000 autoscaled events.
- a viability probe should display, in FIGS. 2 and 3, three characteristics: 1) little or no staining of live cells (i.e., the non-green peaks of the test samples in the first row of FIGS. 2 and 3 should match the green peaks of the unstained controls they overlay as closely as possible) ; 2) fluorescence bimodality comparable to the proportion of live and dead cells in the sample, as indicated in FIGS.
- LS %POS refers to the percentage of cells displaying the low forward scatter characteristic of dead cells
- b %POS refers to percent positivity
- c MC refers to mean channel (unconverted values from a 4-decade logarithmic scale)
- the fluorescence distributions of PI, actin- SAM-FITC, cytokeratin-SAM-FITC, and 7-AAD corresponded to light scatter and trypan blue uptake (data not shown) in the relative proportions of their negative (live cell) and positive (dead cell) peaks; TO-PRO-3's fluorescence distribution remained unimodal throughout the time course.
- actin-SAM-FITC, cytokeratin-SAM- FITC, and 7-AAD suitable for this application, cytokeratin-SAM-FITC exhibits the highest signal-to-noise ratio, remaining distinct from autofluorescence through day 15.
- PI, actin, and cytokeratin positivity remain comparable through day 15 after harvest. This finding is significant, because it suggests that the much greater size of the antibody sandwich complexes, relative to the DNA-specific dyes, does not impede staining.
- PI and TO-PRO-3 fluorescence intensities show the steepest decline, followed by cytokeratin and, in tandem, actin and 7-AAD. Conversely, autofluorescence (green histograms) increases steadily, particularly in the green region of the spectrum, with cell age after harvest.
- FIG. 3 and Table 3 The results of the second time-course study are presented in FIG. 3 and Table 3.
- the formats of both are the same as those of FIG. 2 and Table 2, respectively.
- the light scatter, autofluorescence, and PI staining of unprocessed cells are similar to those in FIG. 2.
- nonspecific staining of live cells was minimal for tubulin-SAM-FITC, and relatively high for PI (in both processed and unprocessed cells) , LDS-751, and EMA.
- LS %P( ->S refers t( 3 the percentage of eel s displaying the low foi • ward scatter characteristic of dead cells
- b %POS refers to percent positivity
- c MC refers to mean channel (unconverted values from a 4-decade logarithmic scale)
- tubulin-SAM-FITC tubulin-SAM-FITC
- EMA tubulin-SAM-FITC
- tubulin-SAM-FITC exhibits the highest signal-to-noi ⁇ e ratio, remaining very distinct from unstained cells through day 15.
- the difference between EMA staining and autofluorescence is approximately the same as the level of nonspecific staining in live cells at day 0.
- PI due to its reversible binding, nor LDS-751, because of its consistently unimodal fluorescence distribution, are acceptable as viability probes for this application.
- the light scatter-based percentages of dead cells in Table 3 are consistently and significantly higher than the fluorescence-based percentages.
- PI in unprocessed cells, tubulin-SAM-FITC, and EMA yield comparable positivity throughout the time course.
- the consistently unimodal distributions of LDS-751 and PI in processed cells make them uninformative here for viability.
- the mean fluorescence intensity of PI in both processed and unprocessed samples declines rapidly over the time course, while that of EMA and tubulin-SAM-FITC diminishes slowly.
- the fluorescence intensity of LDS-751 appears to be cyclical over the time course, while all of the other probes exhibit a linear decrease in fluorescence intensity.
- Healy CG, Kenyon NS, Bolton WE Expression of proliferation associated antigens PCNA, pl45, and Ki67 during cell cycle progression in activated peripheral blood lymphocytes: potential utility in monitoring hematologic malignancies. Cancer Molecular Biology 1:59-70, 1994.
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Abstract
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US18479694A | 1994-01-21 | 1994-01-21 | |
US184796 | 1994-01-21 | ||
PCT/US1995/000513 WO1995020148A1 (fr) | 1994-01-21 | 1995-01-13 | Sondes de viabilite pour isoler, identifier et/ou analyser des cellules |
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JP (1) | JPH09508270A (fr) |
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WO (1) | WO1995020148A1 (fr) |
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US5736129A (en) * | 1995-11-17 | 1998-04-07 | Medenica; Rajko D. | Flow cytometric pharmacosensitivity assay and method of cancer treatment |
US8131053B2 (en) | 1999-01-25 | 2012-03-06 | Amnis Corporation | Detection of circulating tumor cells using imaging flow cytometry |
US8885913B2 (en) | 1999-01-25 | 2014-11-11 | Amnis Corporation | Detection of circulating tumor cells using imaging flow cytometry |
US8406498B2 (en) | 1999-01-25 | 2013-03-26 | Amnis Corporation | Blood and cell analysis using an imaging flow cytometer |
US7450229B2 (en) | 1999-01-25 | 2008-11-11 | Amnis Corporation | Methods for analyzing inter-cellular phenomena |
JP2007526743A (ja) * | 2003-06-06 | 2007-09-20 | ザ・ボード・オブ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・イリノイ | クロマチン分析を利用して細胞の侵襲可能性を評価するための方法 |
US8103080B2 (en) * | 2004-03-16 | 2012-01-24 | Amnis Corporation | Method for imaging and differential analysis of cells |
US8953866B2 (en) | 2004-03-16 | 2015-02-10 | Amnis Corporation | Method for imaging and differential analysis of cells |
CA2598602A1 (fr) | 2004-03-16 | 2005-10-20 | Amnis Corporation | Quantification par image de la translocation moleculaire |
EP3304030B1 (fr) * | 2015-06-01 | 2021-03-17 | Nexcelom Bioscience LLC | Procédés de mesures de viabilité et de nombre de cellules |
JPWO2017169240A1 (ja) * | 2016-03-30 | 2019-02-07 | 富士フイルム株式会社 | 遺伝子解析方法 |
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1995
- 1995-01-13 JP JP7519604A patent/JPH09508270A/ja active Pending
- 1995-01-13 AU AU15669/95A patent/AU1566995A/en not_active Abandoned
- 1995-01-13 EP EP95907435A patent/EP0745217A4/fr not_active Withdrawn
- 1995-01-13 WO PCT/US1995/000513 patent/WO1995020148A1/fr not_active Application Discontinuation
Non-Patent Citations (3)
Title |
---|
BIOLOGICAL ABSTRACTS, vol. 95, Philadelphia, PA, US; abstract no. 174324, XP002063722 & M.C. O'BRIEN ET AL.: "Comparison of cell viability probes compatible with fixation and permeabilization for combined surfaces and intracellular staining in flow cytometry." CYTOMETRY, vol. 19, no. 3, 1995, WASHINGTON DC USA, pages 243-255, * |
See also references of WO9520148A1 * |
W. BOLTON ET AL.: "Simultaneous multiparametric analysis of tissue directed (Cytokeratin), tumor associated (EMA, CEA) and proliferation associated (PCNA, P120) antigens in breast tumor cells." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 33, 1 March 1992, SAN DIEGO CA USA, page 39 XP002063721 * |
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AU1566995A (en) | 1995-08-08 |
EP0745217A4 (fr) | 1998-07-29 |
JPH09508270A (ja) | 1997-08-26 |
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