WO1999045094A1 - Analyse selective de cellules - Google Patents

Analyse selective de cellules Download PDF

Info

Publication number
WO1999045094A1
WO1999045094A1 PCT/US1999/004415 US9904415W WO9945094A1 WO 1999045094 A1 WO1999045094 A1 WO 1999045094A1 US 9904415 W US9904415 W US 9904415W WO 9945094 A1 WO9945094 A1 WO 9945094A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
granulocytes
antibody
interest
subset
Prior art date
Application number
PCT/US1999/004415
Other languages
English (en)
Inventor
Louis A. Kamentsky
Original Assignee
Compucyte 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 Compucyte Corp. filed Critical Compucyte Corp.
Priority to JP2000534626A priority Critical patent/JP2002505441A/ja
Priority to EP99909695A priority patent/EP1066368A4/fr
Priority to CA002321203A priority patent/CA2321203A1/fr
Priority to AU28844/99A priority patent/AU2884499A/en
Publication of WO1999045094A1 publication Critical patent/WO1999045094A1/fr

Links

Classifications

    • 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
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • 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
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G01N15/0618—Investigating concentration of particle suspensions by collecting particles on a support of the filter type
    • G01N15/0625—Optical scan of the deposits
    • 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
    • G01N15/04—Investigating sedimentation of particle suspensions
    • G01N15/042—Investigating sedimentation of particle suspensions by centrifuging and investigating centrifugates
    • 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
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/018—Platelets
    • 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
    • G01N2015/1486—Counting the particles

Definitions

  • This invention relates to devices and methods used in selectively separating cells of varying types and characteristics from each other for analysis and particularly to such methods and devices used with laser scanning cytometers .
  • Flow cytometry instruments measure multiple optical properties of cells as they are made to flow in a cuvette in single file through a light beam, often a laser. As the cell interacts with the light, both fluorescent and scatter emissions are given off that can be measured by photo sensors. If the cells are appropriately reacted prior to measurement, with fluorescent dyes that bind to specific cellular constituents, the amount of fluorescence measured can be directly related to each of a specific cellular constituent, such as DNA, RNA or specific proteins. These dyes can be conjugated by well established techniques to antibodies which can in turn bind to specific proteins in the cell.
  • the resulting luorescence measured by a flow cytometer' s sensors, can be used to quantify the amount of a specific protein per cell or to differentially count one or more types of cell in a heterogeneous cell population such as blood. Additionally, fluorescence dyes can be attached to specific nucleic acid sequences, which in turn, will bind to specific DNA or RNA sequences within the cell's nucleic acids to mark cells with specific genotypes.
  • heterogeneous populations of cells on a slide can be scanned, multiple properties of the cells measured, and the instrument under user direction can relocate cells with specific defined sets of properties for visual observation or photography or to combine data from multiple assays using the location of each cell as the data merge key.
  • constituents of cells can be localized to cell compartments such as the nucleus or cytoplasm and tests such as in-situ hybridizations, in which fluorescent spots are counted, can be performed.
  • cells of interest includes a “cell set (e.g., red blood cells, white blood cells, microorganisms, etc.) containing a subset of cells of interest” (e.g., monocytes) unless otherwise indicated.
  • a cell set e.g., red blood cells, white blood cells, microorganisms, etc.
  • a subset of cells of interest e.g., monocytes
  • the present invention comprises a method and device for adhering cells of interest (or a set of cells containing cells of interest as a subset) to a limited area of a stationary specimen substrate such as a microscope slide, for examination by a laser scanning cytometer.
  • the method of the present invention comprises the steps of: a) treating a heterogeneous cell specimen sample with adhering means which bind to cells of interest or to a selected set of cells including the cells of interest as a subset thereof ; b) adhering cells bound with the adhering means to a limited area substrate by secondary adhering means;
  • steps "a" and “b” in the above method can be replaced with the step of the surface itself being initially provided with the adhering means, whereby the cells of interest, or set of cells containing the cells of interest as a subset, are adhered to the surface in situ, without pre-treatment of the specimen sample. Additionally, irrelevant cells can be separated out with the use of a porous membrane.
  • the analysis and separation is conducted in three separate steps: a) there is an initial separation of a general class of cells from the overall heterogenous sample for adhesion to a surface by means of a relevant reagent ; b) cells of interest which are adhered among other related but irrelevant cells (i.e., the actual cells for which measurements are exclusively contemplated) are marked with a marker reagent (different from the reagent used to adhere the cells) for the identification thereof, separate from the other adhered cells; and c) using one or more reagents for the determination of specific properties (not merely a count) of the marked cells of interest .
  • a slide is provided with a fluid specimen loading conduit, wherein non- impeding gating means, having the secondary adhering means, is
  • Said gating means effects adhering of the cells having the adhering means attached thereto with application of the secondary adhering means in a pre-selected limited gating area.
  • cells of a heterogeneous specimen are reacted with paramagnetic particles that have been coated with material that will bind the particles to an antibody and the antibody is selected so that it will bind to an antigen on the surface of the cell type of interest in the specimen.
  • the heterogeneous specimen is then reacted with the coated paramagnetic particles whereby the particles bind to the cells of interest (or cells of a set including a subset of cells of interest) .
  • Techniques for manufacturing and using such particles in biologic assays are described in U.S. Patents Nos .
  • the cells of interest are adhered to the substrate and thereby separated from irrelevant cells by any of the following procedures : a) the suspension can be made to flow over a magnetic field in a flow chamber to retain cells of interest on the surface.
  • the magnetic material comprises the secondary adhering means (this is similar to procedures described in U.S. Patents Nos. 4,219,411; 4,731,337 and 5,053,344).
  • the cells of interest can be separated from the irrelevant cells by moving a magnetic field through the suspended cells within the stationary fluid, thereby causing the cells to follow the magnetic field to an area within or outside the original stationary suspension (see U.S. Patents Nos. 3,985,649, 3,712,472, 4,272,510, 4,292,920, and 5 , 567 , 326) .
  • Prior art also describes moving particles and cell from one liquid phase to a second liquid phase by a magnetic field (e.g., U.S. Patents Nos. 4,777,145 and 5,279,936).
  • the second method of adhering selected cells to a surface area is to directly deposit antibodies on the substrate surface in the desired limited area.
  • the specimen is made to come into contact with the surface, whereupon antigens on the surface of cells of interest bind to corresponding antibodies on the surface, and irrelevant cells are washed away.
  • Reagents such as cellulose binding domain are commercially available for enhancing antibody to surface binding to achieve antigen capture. This technique has been reviewed by Norden et al, An Experimental Model of Affini ty Cell Separation, Cytometry 16:15- 33 (1994) .
  • a third method of adhering selected cells to a surface area in accordance with the present invention is to use a membrane filter with pores that will selectively allow specific types of cells to pass through the membrane while retaining others.
  • This methodology has been commonly employed for separating blood cells and advantageously for separating leukocytes from red blood cells and platelets, which readily pass through a 5 micron sized pore while retaining leukocytes on the membrane surface.
  • filters are commercially available from numerous suppliers such as Osmotics, Livermore, CA.
  • These membranes may be layered over an absorbent material such as MFS Borosilicate microfiber manufactured by Micro Filtration
  • the absorbent material may be placed below the membrane in a chamber that serves as the waste reservoir.
  • FIGS, la and lb are schematic representations of disposable specimen loading devices constructed in accordance with a preferred embodiment of this disclosure.
  • FIG. 2 is a schematic representation of the optical measurement system used in effecting measurements of the specimen on the devices shown in Figures la and lb.
  • FIG. 3 is a rendering of photo sensor data from a laser scanning cytometer, as the optical measurement system of FIG. 2.
  • FIG. 4 is a schematic depiction of a disposable slide device as used in Figures la and lb, with loading conduit and gated area shown.
  • FIGS. 5a and 5b are data from a laser scanning cytometer of an example of an assay employing an embodiment of the disclosure as set forth in a scattergram and histogram respectively.
  • FIGS. 6a-c are cell scattergrams of various parameters prepared from data from a laser scanning cytometer of an example of an assay of a specimen sample.
  • FIG. 6d is a histogram obtained from the data of FIG. 6a.
  • FIGS. 7 are two histograms obtained from laser scanned cytometer data from platelet bound CD42b-PE-separated and superimposed to obtain a third histogram.
  • FIGS. 8, 9a and 9b are additional cell scattergrams obtained from data obtained from specimens assayed by a laser scanning cytometer.
  • FIGS. lOa-b are histograms of superimposed histograms after assay of two specimens.
  • FIG. 11 is a rendering of an analysis instrument used in the specimen preparation and analysis in accordance with the present invention.
  • FIG. 12a is a dot plot of the position of each granulocyte of a sample on the filter of another embodiment of the present invention.
  • FIG. 12b is a histogram of the laser scanning cytometer
  • CDllb-FITC fluorescence per cell of the sample shown in FIG 12a and a second sample CDllb-FITC fluorescence per cell of the sample shown in FIG 12a and a second sample .
  • FIGS. 13a and 13b are two sets of superimposed histograms of the orange fluorescence values per cell of other specimens. DETAILED DESCRIPTION OF THE INVENTION
  • the method and device of the present invention when combined with use of laser scanning cytometry technology allows the development of an instrumental system that can perform a number of specific cell constituent assays, whereby an unskilled user can obtain rapid results as a point-of-care instrument.
  • a number of clinically important assays are described below, by way of Examples showing use of the present invention, which have been developed using flow cytometry but have not been employed in routine clinical medicine, because of problems inherent with the use of flow cytometry.
  • EXAMPLE I Among assays performable by laser scanning cytometers are tests for activation of specific blood cells caused by sepsis in newborns or adults, which can be detected by measuring the activation of blood granulocytes resulting from interaction of
  • CDllb granulocyte activation is used to determine the condition of patients following angioplasty or stent surgery.
  • the activation of another constituent of blood, platelets as measured by antibodies such as AAC-2 to p-selectin, after their capture to a surface by an anti-platelet antibody or thrombin, and their identification by antibodies such as CD42b is used in an assay for cardiovascular disease.
  • a sensitive assay for early detection of myocardial infarctions (AMI) as well as other vascular pathologies has been devised and tested using flow cytometry (described in U.S. Patent No. 5,503,982) .
  • This assay relies on the discovery that after arterial damage, circulating platelets become activated resulting in p-selectin' s transport to the platelet surface where it binds to blood monocytes.
  • a high ratio of monocytes that are complexed with platelets to uncomplexed monocytes in a patient blood specimen is an indicator of an AMI .
  • leukocytes from whole blood are captured on a small area surface by a CD45 antibody and a fluorescent dye is conjugated to a CD14 antibody which, binds to monocytes, and can be used to identify them.
  • a fluorescent dye is conjugated to a CD14 antibody which, binds to monocytes, and can be used to identify them.
  • EXAMPLE III The method of the present invention is used to assay for drug occupancy on a cell surface.
  • One important clinically useful assay of this type is to measure whether platelet protein GPIIb/IIIa is bound to a drug or is capable of binding to fibrinogen (described in U.S. Patent No. 5,440,020).
  • fibrinogen described in U.S. Patent No. 5,440,020.
  • Such drugs are being developed for treatment of various cardiovascular diseases. It is necessary to monitor the level of these drug's occupancy, since over-treating will lead to other conditions such as stroke. Techniques for performing diagnostic assays of these drugs are disclosed in U.S. Patent No. 5,114,842.
  • blood is activated by an ADP agonist to cause fibrinogen binding to non drug occupied platelets.
  • platelets from the whole blood specimen are captured on a small area substrate surface by a CD42b antibody.
  • a fluorescent dye is conjugated to a CD41 antibody which binds to the GPIIb/IIIa protein in a region separated from the fibrinogen binding region (U.S. Patent No. 5,372,933).
  • the fluorescence of a second fluorescent dye conjugated to a RIBS antibody which binds to fibrinogen when it, in turn, is bound to platelet GPIIb/IIIa is measured.
  • the ratio of the second fluorescence to first fluorescence intensity for each captured platelet is measured and the distribution of this ratio and computed derivatives of it are reported as the assay determination.
  • the method of the present invention may also be used for immunophenotyping, for example, counting T4 cells for AIDS patient monitoring or for diagnosing various leukemias. This is presently the major application for centralized flow cytometry instruments and is clinically employed because results are not required immediately so that an expensive, labor intensive flow cytometer can be employed.
  • the present invention as used with a laser scanning cytometer, is however separately useful because of the very high volume clinical assay of white blood differential counts since laser scanning allows for the relocation of events for visual observation, which is not possible with flow cytometry.
  • leukocytes from whole blood are captured on a small area surface by an antibody such as CD45, a fluorescent dye is conjugated to an antibody such as CD3 that will bind to T lymphocytes and can be used to identify them, and the fluorescence of a second fluorescence dye conjugated to an antibody such as CD4 which binds to T4 helper lymphocytes, is measured.
  • the ratio of the number of CD4 positive cells with second fluorescence to the total number of T lymphocytes is used as the assay determination. This can be extended to many other CD antibodies to detect other cell types. It is contemplated that the methods of the disclosure can be extended to more than detection of one type of cell by using more than two fluorescent dyes conjugated to antibodies.
  • Tube 1 may be a standard vacutainer used to draw blood, with a rubber sealing insert 12 at its top. Tube 1 is inserted into sleeve 13, of the disposable 2, thereby causing tubes 3 and 4 to pierce insert 12.
  • the disposable 2 (preferably made of molded plastic material) contains three chambers 5, 7, and 8 and two ports 6 and 9. Reagents are contained within chamber 5 of the disposable. In one embodiment, in which magnetic particles coated with an antibody are used to cause cells to adhere to a surface, chamber 5 contains coated magnetic particles. In a preferred embodiment, chamber 5 contains two or more antibodies to cellular antigens, each of which is conjugated to a different fluorescent dye. Chamber 5 is connected to tube 4 as well as port 6. The assembly consisting of the specimen tube 1 and disposable 2 is inserted
  • the assay is activated by the user by pressing the enter key of the instrument beginning the test cycle.
  • Port 6 which is connected to a pressure source in the instrument is pressurized then cycled in pressure, to cause the reagent contained in chamber 5 to enter through tube 4 and mix with the specimen in tube 1.
  • the reagent contains two or more antibodies, each conjugated to a different fluorescent dye. In preferred embodiments these are fluorescein isothiocyanate (FITC) and phycoerythrin (PE) which are excited by the wavelength energy of an Argon ion laser. Other dyes such as CY3 , CY5 , APC, CY5.5. or CY7 could be used for excitation by other gas or solid state lasers.
  • the reagent of the preferred embodiment in which cells are adhered by a magnetic field also contains magnetic particles coated with an antibody such as CD45 which will bind to cell surface antigens such as those of white blood cells for CD45.
  • an antibody such as CD45
  • Such particles are commercially available from Perceptive BioSystems, Framingham, MA.
  • the magnetic particles are not part of the reagent.
  • Chamber 7 has dimensions of 50 to 400 microns in depth so that the specimen cells will flow close to the lower surface where there is a magnetic field gradient.
  • the chamber 7 width is set at a value depending on the number of cells necessary to be captured for good statistical results, as cells will be captured perpendicular to the flow.
  • a typical chamber width is about 5 mm.
  • a magnet is placed in the instrument so as to produce a strong magnetic gradient along a line perpendicular to the flow at the area 10. This is accomplished in the Examples by using a rectangular bar magnet with the pole face directed against the
  • the chamber 7, can contain a paramagnetic wire with a triangular cross section embedded in the disposable perpendicular to the flow, so that the wire apex is near the cell flow and the base is in contact with a magnet.
  • the port 6 is then connected to a source of phosphate buffered saline which is made to flow through chamber 5, and into chamber 7, thereby washing irrelevant cells from surface area 10. Waste is collected in chamber 8.
  • Cells are collected over the area 10 (a non-flow impeding gate) defined by the width of chamber 7 and the distribution width characteristics of the magnetic field gradient.
  • the field and chamber dimensions are appropriately adjusted so that the cells of interest are captured as a monolayer for the specimen used.
  • cells can be captured using an antibody coated surface area 10, in place of a magnetic field and magnetic particles.
  • surface area 10 is scanned by a laser scanning cytometer as schematically shown in FIG. 2. The scanning may be preferably done in the same instrument used to capture the cells, or can be done within a second independent instrument.
  • the disposable 26, is held to movable stage 33.
  • the instrument contains a laser 20, such as an Argon ion, HeNe, or solid state laser and the choice will depend on the fluorescent dyes used.
  • the laser beam 21 is reflected by dichroic mirror 22, and computer controlled scanning mirror 23. Such scanning mirrors are commercially available, for example, from General Scanning, Watertown, MA.
  • the beam is passed through lenses 24 and 25, to produce a line scan focused at area 10.
  • the dimensions of this line scan are preferably the width of the cell capture area in scan extent and 5 to 10 microns in beam diameter.
  • the disposable 26 is moved perpendicular to the scan by the stage 33, to raster scan all of area 10. As each cell
  • a third sensor measuring forward angle scatter will be used to find cell data and isolate data belonging to individual cells. This sensor will consist of a light blocking bar and photo sensor placed along the laser light path below disposable 26.
  • FIG. 3 is a representation of data from one sensor as fluorescent emitting cells are scanned where the density at each pixel position is representative of the data value.
  • the data from a typical cell is represented by the pixels 30. Cells are found if a set number of contiguous values above a set threshold are found for any sensor's data. Alternatively a scatter sensor may be used for detecting the presence of and isolating cell data.
  • the computer program first isolates the data belonging to each cell found.
  • contour 31' surrounding the cell at the threshold value .
  • the largest contour of the sensor contours is selected and this is enlarged so that all cell data is used.
  • the pixel values within each contour are then summed.
  • Two additional contours 32' and 33 are constructed by the computer a set distance from contour 31' . For each sensor, the pixel values between these contours are averaged to determine a background level which is subtracted from the corresponding cell sensor
  • the integrated value result is proportional to the fluorescence emission from that cell as detected by each sensor and in turn is proportional to the number of a specific antigen or other constituent bound to fluorescent dye molecules on the cell.
  • Additional information such as the contour area equal to the number of pixels in the contour of each cell or the maximum pixel value in the contour can be computed and may be used to distinguish single cells from cell clusters and distinguish cell states.
  • the integrated values determined as above are used to determine the contents of information displayed to the user.
  • This data may be displayed as two parameter scattergrams, histograms, or numeric values as described in the examples.
  • the result can be displayed on a screen, alphanumerical display, or printed form.
  • EXAMPLE 1 This example describes an experiment to measure the cell capture efficiency for a specific disposable design using magnetic particle capture to adhere cells to a surface in a defined area.
  • Whole blood was diluted 1:1 with phosphate buffered saline (PBS) containing an antibody conjugated to fluorescein isothiocyanate (FITC) .
  • PBS phosphate buffered saline
  • FITC fluorescein isothiocyanate
  • the mouse anti-human antibody CD45 which binds to all leukocytes was used. After an incubation period the cells were washed with PBS and 50 microliters of Perceptive Biosystems magnetic particles conjugated to an anti-mouse antibody were added to 100 microliters of the specimen.
  • a disposable device 40 shown diagrammatically in Fig. 4 was constructed by adhering a 200 ⁇ thick adhesive coated mylar template 41 with a 5 mm wide channel to a standard microscope
  • a standard glass cover slip 43 was adhered to the center of the slide so as to form a 200 ⁇ x 5 mm flow channel 42.
  • a bar magnet was glued below the slide so that one pole was in direct contact with the back surface of the slide and perpendicular to the flow path. In this way two strong magnetic gradients are placed along the specimen flow path and one would expect that maximum capture of magnetically coated cells would occur in proximity to the two edges of the magnet.
  • the specimen was pipetted into the right side of the flow channel and flowed by capillary action through the channel.
  • a cotton wick was placed at the left end of the flow channel and PBS was then pipetted into the flow channel to wash away all irrelevant cells not adhering to the slide. This resulted in the appearance of a line 44 from the excess magnetic particles adhering to the slide surface at a position coinciding with the first edge of the magnet where the magnetic field gradient is strongest .
  • the slide disposable was placed on the microscope stage of a CompuCyte LSC ® , CompuCyte Corporation, Cambridge, MA., laser scanning cytometer and a portion of the cover slip area was scanned to locate the position of each cell found based on detection of FITC fluorescence.
  • the location of each cell can be displayed as a scattergram 50 of Fig. 5a, in which each dot represents a cell at the X and Y coordinates of the axes.
  • the number of cells as a function of position along the flow path can be plotted as a histogram 51 of Fig. 5b, in which numbers of cells at each X position is plotted.
  • EXAMPLE 2 A disposable configured as described in Example 1 was used in all of the remaining examples. In this example, cells are captured with the same antibody used to detect their presence with a first fluorochrome, and a property of the cells is measured using a second antibody and fluorochrome.
  • PE phycoerythrin conjugated mouse anti-human antibody ACC-2 which binds to p-selectin antigen found on the surface of activated platelets.
  • PE fluoresces at a wavelength that can be separated from FITC fluorescence by appropriate optical filters. This example is intended to show the number and extent of activated platelets in a patient blood specimen. After incubation, 50 microliters of anti-mouse antibody conjugated magnetic particles were added to 100 microliters of the mixture, incubated and added to the flow chamber followed by a PBS wash.
  • Figs. 6a-d The position distributions of the detected platelets in the control specimen are shown as a scattergram 60 of Fig. 6a and a histogram 61 of Fig. 6d and are similar to Example 1.
  • the spectral overlap compensated scattergram 63 of Fig. 6c shows the level of activation based on ACC-2 antigen measurement plotted versus
  • CD42b antigen per platelet The data was compensated for filter spectral overlap as practiced in flow cytometry phenotyping. The number of events in each of the four quadrants of display 62 of Fig. 6b were counted and 83% of the platelet events had a relatively high level of ACC-2 p- selectin expression as
  • antibodies can be used to measure cell activation such as antibodies that will bind to GPIIb/IIIa or fibrinogen, as well as p-selectin of platelets. It is also contemplated that this method will be used to measure granulocyte activation using either CDllb or CD64 , or other antibodies expressed on activated granulocytes. It is also understood that the first antibody can be different than the antibody used to capture the cells of interest.
  • EXAMPLE 3 This is an example of application of the preferred embodiment in which cells are captured to a small surface area with a first antibody, a subset of cells is identified with a second antibody, and a characteristic of the cells of the subset is measured with a third antibody.
  • This example demonstrates an assay to determine the percentage of monocytes complexed with platelets. It has been shown in the prior art that cardiovascular injury results in the activation in circulating blood, of platelets which then in turn bind to monocytes. An assay of the ratio of platelet bound to non-platelet bound monoctes is an indicator of cardiovascular injury such as an acute myocardial infarction.
  • Two samples of whole blood from the same individual were obtained. Two micromoler ADP and 5 millimolar GPRP were added to one sample to activate the platelets. Platelets, when activated, bind to some leukocytes including monocytes . Each sample was mixed in a 1:1 ratio with PBS containing three antibodies, the first mouse anti-human CD45 conjugated to Perceptive Biosystems magnetic particles. CD45 binds to a common leukocyte antigen on white blood cells. The second antibody, mouse anti-human CD14 was conjugated to FITC and binds to monocytes. The third antibody, mouse anti-human CD42b, which binds to platelets, was conjugated to the dye phycoerythrin (PE) . The samples were then fixed by adding paraformaldehyde to a 1% concentration to
  • Fig. 7 shows two superimposed histograms of the maximum value within each contour of orange fluorescence from the platelet bound CD42b-PE.
  • the non-activated sample curve 71 is clearly differentiated from the assay result 72 of the activated sample indicating that the preferred embodiment can be used to assay for platelet monocyte complexes in a blood specimen.
  • CD61 and CD 41 may be preferable to CD42b for the assay of this example because the antigen to which CD42b binds may be decreased when platelets are activated.
  • the reagent mixture in this example, contained mouse anti-human CD3 antibody conjugated to PE and mouse anti-human antibody CD4 conjugated to FITC. CD3 binds to antigens found on T lymphocytes while CD4 binds to T4 or helper T lymphocytes.
  • EXAMPLE 5 This example uses the preferred embodiment to demonstrate the common white blood cell differential count.
  • a whole blood specimen was prepared as in examples 3 and 4, using CD45 conjugated to magnetic particles and the monocyte specific mouse anti-human antibody CD14 and the granulocyte specific mouse anti-human antibody CD15.
  • CD14 was conjugated to FITC and CD15 was conjugated to PE.
  • the slide was assayed twice, the first time using green fluorescence data to determine the contour and the second time using orange fluorescence to determine the contour. This was done this way because the CompuCyte LSC does not have the means to simultaneously use two sensors to segment cell data.
  • the resulting data is shown in Figs. 9a and 9b in which the two scattergrams represent data from each of the two assays respectively.
  • the orange fluorescence gated scattergram shows 95% of the cells expressing orange fluorescence are CD15 positive and are granulocytes. Approximately 2920 cells were counted in quadrant region 4. The green fluorescence gated scattergram shows 92% of the cells in quadrant region 1 expressing high levels of CD14 and are monocytes. Approximately 800 cells were counted in region 1. EXAMPLE 6
  • This example uses the preferred embodiment to demonstrate the determination of activation of whole blood granulocytes by the agonist f-Met-Leu-Phe (fMLP) .
  • fMLP f-Met-Leu-Phe
  • bacteria contained in blood, pharmaceuticals, or food may be captured onto a surface using lectins coated to the surface or conjugated to magnetic particles ( See e.g. Payne et al The use of ilmmobolized lectins in the separation of Staphylococcus aureus, Escherichia coli , Listeria and Salmonella spp . From pure cul tures and foods, Journal of Applied Bacteriology 73:41-52 (1992) . Bacteria can be counted by using nucleic acid specific dyes taken up by all bacteria.
  • RNA specific nucleic acid probes can be used to identify bacteria. This is reviewed by Amann et al, Phylogenetic Identification and In Si tu Detection of Individual
  • the surface used to capture the bacteria may be embedded in a growth medium, allowing the live bacteria to remain viable.
  • the capture area may be scanned a second time and the specimen reassayed. Since laser scanning cytometry records the position of each cell, as described in the examples, it is possible to determine changes in fluorescence of any event found. Event contouring can be adjusted to encompass a large enough area so proximate bacteria are within the same contour. If nucleic acid per bacteria, for example, is measured, viable bacteria will have increased amounts of DNA per event measured during the second assay, and their viability indicated.
  • the patterns of located events ( as shown and described in EXAMPLE 1) for the first and second assays can be compared by software to determine the correspondence of each events data in the first and second assays.
  • the corresponding fluorescence values will then indicate bacterial growth by virtue of increases in total fluorescence or size properties between the two assays.
  • This example exemplifies the embodiment in which all leukocytes from a whole blood specimen are separated from red blood cells by a membrane with 5 micron pores.
  • the example demonstrates an assay for determination of the activation of whole blood granulocytes by the agonist f-Met-Leu-Phe (fMLP) .
  • fMLP f-Met-Leu-Phe
  • Two samples of the same blood specimen were obtained. 10M fMLP was added to one sample at 37°C, and the sample was incubated for 10 minutes to cause activation of the sample's granulocytes .
  • Each sample was then separately mixed in a 1:1 ratio with PBS containing two antibodies.
  • a first antibody, mouse anti-human CD15 that binds to granulocytes was conjugated to the dye phycoerythrin (PE) .
  • PE dye phycoerythrin
  • the second antibody, mouse anti- human CDllb that binds to an activation antigen of granulocytes was conjugated to the dye fluorescein isothiocyanate (FITC) .
  • FITC dye fluorescein isothiocyanate
  • 100 microliters of each mixture was pipetted into the center of an Osmotics (Catalog no. 10572) , 5 micron pore size, 13 mm membrane filter placed over MFS Borosilicate microfiber absorbent material (Micro Filtration Systems, Dublin, CA) . This was immediately followed by the pipetting of 200 microliters of PBS onto the membrane center surface. Each filter was removed, placed on a microscope slide and covered with a slip and assayed on the laser scanning cytometer.
  • Granulocytes were detected and contoured based on orange fluorescence from the PE bound to the granulocytes .
  • the green FITC fluorescence resulting from the binding of CDllb to the granulocyte activation antigen was measured for every contoured event. Since the LSC cytometer is capable of recording and displaying the position of each found event, it was used for such purpose.
  • the positions of each granulocyte on the filter, for the first specimen, is displayed as a dot plot 101 of Fig.
  • the histogram display was divided into two regions by the boundary line 105 and the counts of cells on the right (positive CD lib) side of the boundary was determined for each of the unstimulated and stimulated specimen. For these assays the unstimulated count was 87 of 15,882 or 5.1% of the total cells counted and the stimulated specimen's count was 7342 of 9043 or 81.2%.
  • EXAMPLE 8 This is an example of an application of the third embodiment in which cells are captured on a membrane of small surface area, wherein a subset of cells is identified with a first antibody, and wherein a characteristic of the cells of the subset is measured with a second antibody.
  • the example illustrates an assay of determination of the percentage of monocytes complexed with platelets.
  • cardiovascular injury results in the activation, in circulating blood, of platelets which in turn bind to monocytes.
  • An assay of the ratio of platelet bound to non-platelet bound monocytes is an indicator of cardiovascular injury, such as an acute myocardial infarction.
  • Figs. 13a and 13b show two sets of superimposed histograms of the orange fluorescence values per cell, the histograms 106 from the platelet bound CD42b-PE specimen, and the histograms 109 from the platelet bound CD62-PE specimen.
  • the non-activated sample curve 1077 is clearly differentiated from the assay result 108 of the activated sample as is the non-activated curve 110, differentiated from activated curve 111.
  • the histogram displays were divided into two regions by the boundary lines 109a and 112 respectively and the counts of cells on the right side of the boundary consisting of complexed monocytes and platelets (positive CD42b or CD62) was determined for each of the unstimulated and stimulated specimens. For these assays the unstimulated count using CD42b was 111 of 2408 or 4.6% of the total cells counted and the stimulated specimen's count was 1681 of 2267 or 74.2%.
  • CD62 Using CD62 it was 448 of 6053 or 7.4% of the total cells counted and the stimulated specimen's count was 3614 of 4616 or 78.3%.
  • This embodiment for effecting the requisite separation is a preferred embodiment, as one which can also be used to assay for platelet monocyte complexes in a blood specimen.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Dispersion Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

L'invention concerne un procédé et un dispositif d'analyse par balayage laser d'une petite partie sélectionnée de cellules de prélèvements dans une zone fixe et très localisée d'une lamelle porte-objet, ces cellules étant d'abord spécifiquement sélectionnées pour présenter un intérêt au balayage. Les prélèvements peuvent être prétraités avec une substance adhésive spécifiquement adaptée aux caractéristiques des cellules sélectionnées, les cellules spécifiques étant ainsi rendues adhésives. La lamelle porte-objet est ensuite localement pourvue d'une matière adhésive ou d'un dispositif adhésif à action conjointe, ou d'un dispositif de filtrage se trouvant dans une position de barrière non gênante, un nombre statistiquement important de cellules sélectionnées n'atteignant pas la barrière ou adhérant à cette dernière alors que le reste de cellules non sélectionnées est retiré de la barrière. La barrière est ensuite balayée par le cytomètre à balayage laser avec une efficacité accrue. Les matières adhésives recouvrent la surface de la zone de barrière ou alors elles comprennent des particules à sensibilité magnétique. Dans le dernier mode de réalisation, un aimant localisé est disposé à côté de la zone de barrière pour faire adhérer des cellules sélectionnées magnétisées à la zone de barrière. Les cellules intéressantes peuvent constituer une classe ou un ensemble de cellules comprenant un sous-ensemble (par exemple des leucocytes et des monocytes), des caractéristiques du sous-ensemble étant intéressantes à analyser. Ainsi, on utilise des réactifs initiaux pour l'adhérence et des réactifs supplémentaires pour le marquage du sous-ensemble et l'analyse des caractéristiques voulues.
PCT/US1999/004415 1998-03-02 1999-03-01 Analyse selective de cellules WO1999045094A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2000534626A JP2002505441A (ja) 1998-03-02 1999-03-01 選択的細胞分析
EP99909695A EP1066368A4 (fr) 1998-03-02 1999-03-01 Analyse selective de cellules
CA002321203A CA2321203A1 (fr) 1998-03-02 1999-03-01 Analyse selective de cellules
AU28844/99A AU2884499A (en) 1998-03-02 1999-03-01 Selective cell analysis

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US3296498A 1998-03-02 1998-03-02
US09/032,964 1998-03-02
US9425698A 1998-06-09 1998-06-09
US09/094,256 1998-06-09

Publications (1)

Publication Number Publication Date
WO1999045094A1 true WO1999045094A1 (fr) 1999-09-10

Family

ID=26709111

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/004415 WO1999045094A1 (fr) 1998-03-02 1999-03-01 Analyse selective de cellules

Country Status (5)

Country Link
EP (1) EP1066368A4 (fr)
JP (1) JP2002505441A (fr)
AU (1) AU2884499A (fr)
CA (1) CA2321203A1 (fr)
WO (1) WO1999045094A1 (fr)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001033190A2 (fr) * 1999-11-04 2001-05-10 Arcturus Engineering, Inc. Microdissection automatique par piegeage laser
WO2002003052A2 (fr) * 2000-07-05 2002-01-10 Board Of Regents, The University Of Texas System Cytologie a balayage laser a capture d'images numeriques
US6665060B1 (en) 1999-10-29 2003-12-16 Cytyc Corporation Cytological imaging system and method
WO2006017811A3 (fr) * 2004-08-06 2006-06-15 Compucyte Corp Absorption de lumiere monochromatique a couleurs multiples et quantification d'absorption de lumiere dans un echantillon colore
EP2041563A1 (fr) * 2006-07-17 2009-04-01 Hemocue AB Numeration de thrombocytes
WO2010058373A1 (fr) * 2008-11-24 2010-05-27 Koninklijke Philips Electronics N.V. Procédé et appareil pour analyse par filtre rapide d'échantillons fluides
GB2476663A (en) * 2009-12-31 2011-07-06 Blood Analysis Ltd Detection of microorganisms
WO2012012803A3 (fr) * 2010-07-23 2012-11-01 Advanced Cell Technology, Inc. Procédés de détection de sous-populations rares de cellules et compositions de cellules très purifiées
US8318445B2 (en) * 2008-01-07 2012-11-27 Luminex Corporation Immunomagnetic capture and imaging of biological targets
US9040770B2 (en) 2004-01-23 2015-05-26 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
US9040039B2 (en) 2004-01-23 2015-05-26 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
CN106198963A (zh) * 2016-08-31 2016-12-07 上海美吉生物医药科技有限公司 一种用于捕获白细胞的免疫磁珠及其制备方法
US10077424B2 (en) 2007-10-12 2018-09-18 Astellas Institute For Regenerative Medicine Methods of producing RPE cells and compositions of RPE cells
US10156501B2 (en) 2001-11-05 2018-12-18 Life Technologies Corporation Automated microdissection instrument for determining a location of a laser beam projection on a worksurface area
US10485829B2 (en) 2009-11-17 2019-11-26 Astellas Institute For Regenerative Medicine Methods of producing human RPE cells and pharmaceutical preparations of human RPE cells
WO2023125942A1 (fr) * 2021-12-31 2023-07-06 深圳迈瑞生物医疗电子股份有限公司 Analyseur de cellules sanguines, procédé et utilisation de paramètre de marqueur d'infection
WO2023107380A3 (fr) * 2021-12-06 2023-08-03 Intellifoods Labs, Llc Système de microscope électronique combinant la fluorescence et le balayage pour la détection d'agents pathogènes

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6350613B1 (en) * 1998-03-07 2002-02-26 Belton Dickinson & Co. Determination of white blood cell differential and reticulocyte counts
US7588905B2 (en) * 2003-04-16 2009-09-15 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Identification and monitoring of systemic lupus erythematosus

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5053344A (en) * 1987-08-04 1991-10-01 Cleveland Clinic Foundation Magnetic field separation and analysis system
US5100777A (en) * 1987-04-27 1992-03-31 Tanox Biosystems, Inc. Antibody matrix device and method for evaluating immune status
US5107422A (en) * 1989-10-02 1992-04-21 Kamentsky Louis A Method and apparatus for measuring multiple optical properties of biological specimens
US5602042A (en) * 1994-04-14 1997-02-11 Cytyc Corporation Method and apparatus for magnetically separating biological particles from a mixture
US5660990A (en) * 1995-08-18 1997-08-26 Immunivest Corporation Surface immobilization of magnetically collected materials
US5663057A (en) * 1994-11-17 1997-09-02 Chemunex Process for rapid and ultrasensitive detection and counting of microorganisms by fluorescence

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5100777A (en) * 1987-04-27 1992-03-31 Tanox Biosystems, Inc. Antibody matrix device and method for evaluating immune status
US5053344A (en) * 1987-08-04 1991-10-01 Cleveland Clinic Foundation Magnetic field separation and analysis system
US5107422A (en) * 1989-10-02 1992-04-21 Kamentsky Louis A Method and apparatus for measuring multiple optical properties of biological specimens
US5602042A (en) * 1994-04-14 1997-02-11 Cytyc Corporation Method and apparatus for magnetically separating biological particles from a mixture
US5663057A (en) * 1994-11-17 1997-09-02 Chemunex Process for rapid and ultrasensitive detection and counting of microorganisms by fluorescence
US5660990A (en) * 1995-08-18 1997-08-26 Immunivest Corporation Surface immobilization of magnetically collected materials

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAMENTSKY L. A., ET AL.: "SLIDE-BASED LASER SCANNING CYTOMETRY.", ACTA CYTOLOGICA., INTERNATIONAL ACADEMY OF CYTOLOGY, CHICAGO, IL,, US, vol. 41., no. 01., 1 January 1997 (1997-01-01), US, pages 123 - 143., XP002920835, ISSN: 0001-5547 *
See also references of EP1066368A4 *

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6665060B1 (en) 1999-10-29 2003-12-16 Cytyc Corporation Cytological imaging system and method
US7411664B2 (en) 1999-10-29 2008-08-12 Cytyc Corporation Cytological imaging system and method
WO2001033190A2 (fr) * 1999-11-04 2001-05-10 Arcturus Engineering, Inc. Microdissection automatique par piegeage laser
WO2001033190A3 (fr) * 1999-11-04 2002-05-10 Arcturus Eng Inc Microdissection automatique par piegeage laser
WO2002003052A2 (fr) * 2000-07-05 2002-01-10 Board Of Regents, The University Of Texas System Cytologie a balayage laser a capture d'images numeriques
WO2002003052A3 (fr) * 2000-07-05 2002-04-18 Univ Texas Cytologie a balayage laser a capture d'images numeriques
US6656683B1 (en) 2000-07-05 2003-12-02 Board Of Regents, The University Of Texas System Laser scanning cytology with digital image capture
US10156501B2 (en) 2001-11-05 2018-12-18 Life Technologies Corporation Automated microdissection instrument for determining a location of a laser beam projection on a worksurface area
US9650607B2 (en) 2004-01-23 2017-05-16 Astellas Institute For Regenerative Medicine Modalities for the treatment of degenerative diseases of the retina
US9040038B2 (en) 2004-01-23 2015-05-26 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
US9730962B2 (en) 2004-01-23 2017-08-15 Astellas Institute For Regenerative Medicine Modalities for the treatment of degenerative diseases of the retina
US9649340B2 (en) 2004-01-23 2017-05-16 Astellas Institute For Regenerative Medicine Methods for producing enriched populations of human retinal pigment epithelium cells
US9562217B2 (en) 2004-01-23 2017-02-07 Astellas Institute For Regenerative Medicine Modalities for the treatment of degenerative diseases of the retina
US9193950B2 (en) 2004-01-23 2015-11-24 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
US9181524B2 (en) 2004-01-23 2015-11-10 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
US9080150B2 (en) 2004-01-23 2015-07-14 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
US9045732B2 (en) 2004-01-23 2015-06-02 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
US9040770B2 (en) 2004-01-23 2015-05-26 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
US9040039B2 (en) 2004-01-23 2015-05-26 Ocata Therapeutics, Inc. Modalities for the treatment of degenerative diseases of the retina
WO2006017811A3 (fr) * 2004-08-06 2006-06-15 Compucyte Corp Absorption de lumiere monochromatique a couleurs multiples et quantification d'absorption de lumiere dans un echantillon colore
US7468796B2 (en) 2004-08-06 2008-12-23 Compucyte Corporation Multiple-color monochromatic light absorption and quantification of light absorption in a stained sample
US11703428B2 (en) 2004-09-25 2023-07-18 Life Technologies Corporation Automated microdissection instrument and method for processing a biological sample
US11175203B2 (en) 2004-09-25 2021-11-16 Life Technologies Corporation Automated microdissection instrument using tracking information
US10605706B2 (en) 2004-09-25 2020-03-31 Life Technologies Corporation Automated microdissection instrument with controlled focusing during movement of a laser beam across a tissue sample
EP2041563A4 (fr) * 2006-07-17 2013-09-18 Hemocue Ab Numeration de thrombocytes
EP2041563A1 (fr) * 2006-07-17 2009-04-01 Hemocue AB Numeration de thrombocytes
US10077424B2 (en) 2007-10-12 2018-09-18 Astellas Institute For Regenerative Medicine Methods of producing RPE cells and compositions of RPE cells
US8318445B2 (en) * 2008-01-07 2012-11-27 Luminex Corporation Immunomagnetic capture and imaging of biological targets
WO2010058373A1 (fr) * 2008-11-24 2010-05-27 Koninklijke Philips Electronics N.V. Procédé et appareil pour analyse par filtre rapide d'échantillons fluides
US8991270B2 (en) 2008-11-24 2015-03-31 Koninklijke Philips N.V. Method and apparatus for rapid filter analysis of fluid samples
CN102224407A (zh) * 2008-11-24 2011-10-19 皇家飞利浦电子股份有限公司 用于对流体样本快速过滤分析的方法和仪器
US10485829B2 (en) 2009-11-17 2019-11-26 Astellas Institute For Regenerative Medicine Methods of producing human RPE cells and pharmaceutical preparations of human RPE cells
US11850261B2 (en) 2009-11-17 2023-12-26 Astellas Institute For Regenerative Medicine Methods of producing human RPE cells and pharmaceutical preparations of human RPE cells
GB2476663A (en) * 2009-12-31 2011-07-06 Blood Analysis Ltd Detection of microorganisms
WO2012012803A3 (fr) * 2010-07-23 2012-11-01 Advanced Cell Technology, Inc. Procédés de détection de sous-populations rares de cellules et compositions de cellules très purifiées
US11739366B2 (en) 2010-07-23 2023-08-29 Astellas Institute For Regenerative Medicine Methods for detection of rare subpopulations of cells and highly purified compositions of cells
CN106198963A (zh) * 2016-08-31 2016-12-07 上海美吉生物医药科技有限公司 一种用于捕获白细胞的免疫磁珠及其制备方法
WO2023107380A3 (fr) * 2021-12-06 2023-08-03 Intellifoods Labs, Llc Système de microscope électronique combinant la fluorescence et le balayage pour la détection d'agents pathogènes
WO2023125942A1 (fr) * 2021-12-31 2023-07-06 深圳迈瑞生物医疗电子股份有限公司 Analyseur de cellules sanguines, procédé et utilisation de paramètre de marqueur d'infection

Also Published As

Publication number Publication date
EP1066368A1 (fr) 2001-01-10
EP1066368A4 (fr) 2002-08-21
JP2002505441A (ja) 2002-02-19
CA2321203A1 (fr) 1999-09-10
AU2884499A (en) 1999-09-20

Similar Documents

Publication Publication Date Title
EP1066368A1 (fr) Analyse selective de cellules
US5464752A (en) Automated analyzer for screening cells or formed bodies for enumeration of populations expressing selected characteristics
US6251615B1 (en) Cell analysis methods
US20170336386A1 (en) Method and apparatus for analyzing individual cells or particulates using fluorescent quenching and/or bleaching
US20020028471A1 (en) Cell analysis methods and apparatus
JPH0593724A (ja) 特異性化血液細胞の亜綱の自動化された同定および計数装置
US6159740A (en) Method and apparatus for screening obscured or partially obscured cells
CA2518677A1 (fr) Procedes et appareil utilises pour la detection et la quantification de divers types de cellules et utilisation d'un bio-disque optique pour leur mise en oeuvre
US5260192A (en) Method and apparatus for screening cells or formed bodies with populations expressing selected characteristics utilizing at least one sensing parameter
US5231005A (en) Method and apparatus for screening cells or formed bodies with populations expressing selected characteristics
EP0381669B1 (fr) Procede de selection de cellules de corps formes pour enumeration de populations exprimant des caracteristiques selectionnees
Gilman-Sachs Flow cytometry
AU635020B2 (en) Method and apparatus for screening cells or formed bodies with populations expressing selected characteristics utilizing at least one sensing parameter
EP0472522B1 (fr) Procede de triage de cellules ou de corps formes a l'aide de populations exprimant des caracteristiques selectionnees
EP1057004A1 (fr) Procedes et appareil d'analyse de cellules
JPH06505099A (ja) 偏光光散乱手法を用いて微視的細胞をスクリーニングする方法および装置
Park et al. Single-color multitarget flow cytometry using monoclonal antibodies labeled with different intensities of the same fluorochrome
JP2023532780A (ja) 非癌細胞の代謝活性を評価する方法
Steinkamp et al. Automated analysis and separation of cells from the respiratory tract: preliminary characterization studies in hamsters.
JPS58172549A (ja) 血清学的に選定された細胞個体群の計数方法
AlQahtani Application and Disadvantages of Flow Cytometry in Hematopathology: A Brief Review

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2321203

Country of ref document: CA

Ref country code: CA

Ref document number: 2321203

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 534626

Kind code of ref document: A

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: KR

WWE Wipo information: entry into national phase

Ref document number: 1999909695

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1999909695

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1999909695

Country of ref document: EP