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
• • 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
  • G01N2015/016—White blood 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
  • G01N15/1434—Optical arrangements
  • G01N2015/1447—Spatial selection

Definitions

• the present invention relates to particle discrimination by light scattering, and more particularly to a flow cytometer and method therefore that discriminates particles employing a high numerical aperture.
• Numerical aperture is defined as the refractive index of the medium through which light is collected multiplied by the sine value of one- half of the angle of light collection.
• the discrimination of particles is useful in numerous clinical assays including ascertaining the types and numerical quantity of cells in blood, ascertaining invasive particles in a fluid sample, such as bacteria and virus, and quantifying the density and volume of cells in a fluid sample.
• the '497 Patent discloses a flow cell 2 through which cells from, for example, blood or the like, flow substantially one by one therethrough.
• a laser input 4 emits a polarized beam of laser light that is oriented substantially orthogonally to the flow of blood cell through flow cell 2 such that the polarized laser light impinges upon the blood cells as they pass through flow cell 2.
• polarized it is meant that the plane of the electric field oscillization of the laser light is uniform.
• An optical lens 6 has an aperture which limits the cone of scattered light from the blood cells that can be collected to 72° or less. The central axis of the cone of lens 6 is 90° to both the path of the polarized laser light and the flow of blood cells through flow cell 2.
• the scattered light emulating from lens 6 is columnated in a matter known in the art.
• the scattered light now has a mixed polarization that is characteristic of the cell type.
• the light next passes through a beam splitter 8 that divides the light into two separate beams.
• a first light beam, substantially concentric with the light beam that originally eminated from lens 6, passes through first polarization analyzer 10.
• Polarization analyzer 10 is configured to pass therethrough only polarized light having a vector the same as the original laser light.
• the second beam eminating from beam splitter 8 is oriented substantially perpendicular to the orientation of the first beam eminating from beam splitter 8. This second beam enters second polarization analyzer 12.
• Second polarization analyzer 12 is configured to pass therethrough only light having a polarization vector substantially orthogonal to the polarization vector of the other beam from beam splitter 8 that passed through first polarization analyzer 10.
• the beams that pass through first polarization analyzer 10 and second polarization 12 enter polarized detector 14 and depolarized light detector 16, respectively.
• FIG. 4 is a graphical representation having the output of polarized light detector 14 as one axis and the output of depolarized light detector 16 as the axis. While the above invention does provide some useful data regarding leukocytes, and more specifically eosinophils, as shown in FIGS.
• the cluster points within the eosinophil cluster are quite condensed.
• the dense nature of the points within the eosinophil cluster results in difficulty for the computer software programs that ascertain and identify clusters to accurately identify eosinophil clusters.
• this prior art configuration requires expensive optical devices such as photo multiplier tubes, and lens 6, first polarization amplifier 10 and second polarization amplifier 12.
• the high numerical aperture flow cytometer of the present invention includes a flow cell and a laser input.
• the laser input emits a beam of light that is oriented substantially orthoganilly to the flow of blood cells through the flow cell such that laser light impinges upon the blood cells as they pass through the flow cell.
• the laser light emitted by the laser input need not be polarized for analysis of the cells according to the present invention.
• a portion of the beam from the laser input that impinges upon the blood cells in the flow cell is scattered at a substantially right angle to the beam of laser input ("right angle scatter light").
• a second portion of the beam from the laser input that impinges upon the cells in the flow cell is scattered at a much lower angle than 90°.
• This scatter is termed "low angle forward scatter light” and has an angle of from about 2° to about 5° from the orientation of the original beam from laser input.
• a right angle scatter light detector is oriented to receive the previously mentioned right angle scatter light.
• the right angle scatter light detector is preferably located about 2 millimeters from the blood cells in the flow cell.
• An important aspect of the present invention is that, at the distance of about 2 millimeters from the blood cells, the right angle scatter light detector collects a cone of scattered light of at least 100° or greater, and preferably 130° or greater. It is this larger light cone value over the prior art light cone of about 72° that results in the greater cluster separation in the present invention due to the larger signal gathered. In contrast, the smaller 72° cone of the prior art results in missed signals and lesser cluster separation.
• a low angle forward scatter light detector is oriented to capture the previously mentioned low angle forward scatter light oriented at about 2° to about 5° from the beam of the laser input.
• both right angle scatter light detector and low angle forward scatter light detector are employed in order to produce a 2- dimensional cytrogram.
• only right angle scatter light detector is employed, low angle forward scatter light detector is not employed, and characterization of eosinophils is possible.
• FIG. 1 is a schematic representation of the electro-optical components of prior art
• FIG. 2 is a schematic representation of the electro-optical components of the present invention
• FIG. 3 is a block diagram of the electronic processing components of the present invention.
• FIG. 4 is a graphical representation of the separation of eosinophils and other white blood cell components based on light scatter in the prior art
• FIG. 5 is a graphical representation of the separation of eosinophils and other white blood cell components based on light scatter in the present invention
• FIG. 6 A is a graphical representation of 2% canine eosinophil data employing the prior art
• FIG. 6B is a graphical representation of 2% canine eosinophil data employing the present invention.
• FIG. 7A is a graphical representation of 8% canine eosinophil data employing the prior art
• FIG. 7B is a graphical representation of 8% canine eosinophil data employing the present invention
• FIG. 8 A is a graphical representation of 10% canine eosinophil data employing the 5 prior art
• FIG. 8B is a graphical representation of 10% canine eosinophil data employing the present invention.
• FIG. 9 A is a graphical representation of human eosinophil data employing the prior 0 art.
• FIG. 9B is a graphical representation of human eosinophil data employing the present invention.
• the high numerical aperture flow cytometer of the present invention includes a flow cell 18, which is preferably a quartz flow cell manufactured by Opco Laboratories of Fitchburg, Massachusetts.
• flow cell 18 has a flow length of about 1 centimeter and a cross section of 4 millimeter by 4 millimeter. Cells from, for example, blood or the like, flow substantially one by one through flow cell 18 during
• Laser input 20 emits a beam of light that is oriented substantially orthoganilly to the flow of blood cells through flow cell 18 such that laser light impinges upon the blood cells as they pass through flow cell 18. Unlike the prior art, the laser light emitted by laser input 20 need not be polarized for analysis of the cells according to the present invention. .
• Laser input 20 maybe for example a 635 manometer semiconductor diode laser with an output power of 10 miliwatts, model No. HL6320G manufactured by Hatachi and available from Thor Labs, Inc. of Newton, New Jersey.
• a portion of the beam from laser input 20 that impinges upon the blood cells in flow cell 18 is scattered at a substantially right angle to the beam of laser input 20 ("right angle scatter light").
• right angle scatter light detector 22 is oriented to receive the previously mentioned right angle scatter light.
• Right angle scatter light detector is preferably located about 2 millimeters from the blood cells in the flow cell 18.
• An important aspect of the present invention is that, at the distance of about 2 millimeters from the blood cells, right angle scatter light detector 22 collects a cone of scattered light of at least 100° or greater, and preferably 130° or greater. It is this larger light cone value over the prior art light cone of about 72° that results in the greater cluster separation in the present invention due to the larger signal gathered. In contrast, the smaller 72° cone of the prior art results in missed signals and lesser cluster separation.
• Low angle forward scatter light detector 24 is oriented to capture the previously mentioned low angled forward scatter light oriented at about 2° to about 5° from the beam of laser input 20.
• Both right angle scatter light detector 22 and low angle forward scatter light detector 24 can be, for example, silicone PIN photodiodes Model No. S5106PIN manufactured by Hamamatsu Corp. of Bridgewater, New Jersey.
• both right angle scatter light detector 22 and low angle forward scatter light detector 24 are employed in order to produce a 2- dimensional cytrogram.
• only right angle scatter light detector 22 is employed, low angle forward scatter light detector 24 is not employed, and characterization of eosinophils is possible.
• the electrical outputs from right angle scatter light detector 22 and low angle forward scatter light detector 24, which may be in voltage or current form, for example, are amplified by preamplifier 26 and then sent to signal processor 28.
• Signal processor 28 measures the area under the voltage or current curve, or measures the peak of the voltage or current curve, received from right angle light scatter detector 22 and/or low 5 angle forward scatter light detector 24.
• the data from signal processor 28 is converted by analog to digital converter 30.
• the digital data is next processed by central processing unit 32 based on software programs to display the data in graphical representation on display 34. It will be readily apparent to those skilled in the art that the signal amplification, processing, conversion and display can be accomplished by many well ⁇ o known methods, including but not limited to those disclosed in Practical Flow Cytometry
• FIG. 5 the output of the data from the flow cytometer of the present invention is shown.
• FIG. 5 has the output of right angle scatter light detector 22 as one axis and the output of low angle forward scatter light detector 24 as the other axis.
• Eosinophils are located to the right of the software threshold line and, as shown in FIGS.
• FIGS. 6A, 6B, 7A, 7A, 7B, 8A, 8B, 9A and 9B graphical representations of leukocyte identification is shown, with specific reference to eosinophil identification.
• the data of FIGS. 6 A, 7 A, 8 A, and 9 A was employed using the apparatus of the present invention.
• the term R2 denotes primarily
• FIGS. 6B, 7B, 8B, and 9B pertain to data employing an apparatus substantially disclosed in US Patent No. 5,017,497.
• Whole blood samples of either canine or human blood were prepared as follows before analyzing with the apparatus of present invention or the prior art. The whole blood sample was diluted 10 to
• phosphate buffered saline treated whole blood sample was mixed with 1 ,200 microliters of a lysing solution.
• the lysing solution consisted of 8.3 grams of ammonium chloride, 1 gram of potassium bicarbonate, 0.37 grams tetrasodium EDTA per liter of lysing solution.
• the whole blood sample was lysed for 20 minutes to one-half of an hour. It will be readily understood by those skilled in the art that lyse time can readily be reduced to between 30 seconds and one minute.
• FIGS. 6A, 7A, 8 A and 9 ⁇ A good correlation exists between the eosinophil of the present invention of FIGS. 6A, 7A, 8 A and 9 ⁇ with the eosinophil data of the DEPOL/ORTHOGONAL graphical representation of the prior art as shown in FIGS. 6B, 7B, 8B and 9B. More specifically, regarding FIGS. 6A and 6B, the eosinophil value for the present invention is 2.1% and for the prior art is 2.0%. Regarding FIGS. 7A and 7B, the eosinophil data for the present invention is 7.6% and for the prior art is 8.2%. Regarding FIGS. 8A and 8B the eosinophil data for the present invention is 13.1% and for the prior art is 9.8%. Regarding FIGS.
• FIGS. 6A, 7 A, 8 A and 9 A an eosinophil cluster is present at R5.
• the SIZE/COMPLEXITY graphical representation shows no eosinophil cluster, while the graphical representation of FIG. 9B does show a cluster.
• FIGS. 6A, 7 A, 8 A and 9A show a marked decreased density or concentration of the cluster points within the eosinophil clusters.
• the separation of these cluster points allows the software programs that locate and identify different clusters to more readily locate and identify the clusters produced by the apparatus and method of the present invention compared to those of the prior art.

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• Chemical & Material Sciences (AREA)
• Dispersion Chemistry (AREA)
• Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 2000
  • 2000-02-18 CA CA002329031A patent/CA2329031C/en not_active Expired - Fee Related
  • 2000-02-18 JP JP2000600079A patent/JP2002537557A/ja active Pending
  • 2000-02-18 EP EP00914609A patent/EP1095256A2/en not_active Ceased
  • 2000-02-18 WO PCT/US2000/004069 patent/WO2000049387A2/en not_active Ceased
  • 2000-02-18 AU AU35976/00A patent/AU768616C/en not_active Ceased

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