AU768616B2 - High numerical aperture flow cytometer and method of using same - Google Patents
High numerical aperture flow cytometer and method of using same Download PDFInfo
- Publication number
- AU768616B2 AU768616B2 AU35976/00A AU3597600A AU768616B2 AU 768616 B2 AU768616 B2 AU 768616B2 AU 35976/00 A AU35976/00 A AU 35976/00A AU 3597600 A AU3597600 A AU 3597600A AU 768616 B2 AU768616 B2 AU 768616B2
- Authority
- AU
- Australia
- Prior art keywords
- right angle
- light
- scattered light
- flow cell
- flow
- 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.)
- Ceased
Links
- 238000000034 method Methods 0.000 title claims description 21
- 210000004027 cell Anatomy 0.000 claims description 78
- 210000003979 eosinophil Anatomy 0.000 claims description 35
- 210000000601 blood cell Anatomy 0.000 claims description 22
- 239000002245 particle Substances 0.000 claims description 17
- 210000000265 leukocyte Anatomy 0.000 claims description 10
- 238000000149 argon plasma sintering Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 3
- 241000700605 Viruses Species 0.000 claims description 2
- 239000010453 quartz Substances 0.000 claims description 2
- 239000004065 semiconductor Substances 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims 1
- 244000046052 Phaseolus vulgaris Species 0.000 claims 1
- 230000010287 polarization Effects 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 241000282465 Canis Species 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 230000002934 lysing effect Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000028161 membrane depolarization Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
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
Landscapes
- 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)
Description
1 HIGH NUMERICAL APERTURE FLOW CYTOMETER AND METHOD OF USING SAME Field of the Invention The present invention relates to particle discrimination by light scattering, and more particularly to a flow cytometer and method therefor 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 onehalf of the angle of light collection.
Background of the Invention l0 The discrimination of particles is useful in numerous clinical assays including ascertaining the types and numerical quantity of cells in blood, ascertaining invasive particles of a fluid sample, such as bacteria and virus, and quantifying the density and volume of cells in a fluid sample.
One method of the above is disclosed in U.S. Patent No. 5,017,497 issued to de Grooth et al. Referring to FIG. 1, 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. By "polarized" it is meant that the plane of the electric field oscillation of the laser light is uniform. An ooptical lens 6 has an aperture which limits the cone of scattered light from the blood cells i °.that can be collected to 720 or less. The central axis of the cone of lens 6 is 900 to both the path of the polarized laser light and the flow of blood cells through flow cell 2. The scattered light emanating from lens 6 is collimated in a manner known in the art. The S 25 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 emanated 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 30 original laser light. The second beam emanating from beam splitter 8 is oriented substantially perpendicular to the orientation of the first beam emanating from beam splitter 8. This second beam enters second polarization analyzer 12. Second polarization o 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 [R:\LIBZZ05880.doc:mrr II~~I *(UI IC IMI II*III~ Y I- IYIY-YY L -nl* nUIIU I I1I 1 II1 I Il IIIIIYIII* DY-IY NI l 3i.*~Yb.l-m~(--*-YI-IIPIIII I UII Y-LIIRIIU I- ~.InlU -UI I ~Ci 2 polarization analyzer 10 and second polarization 12 enter polarized detector 14 and depolarized light detector 16, respectively. The ratio of the outputs of polarized light detector 14 and depolarized light detector 16, based on intensity, provide the depolarization ratio.
As shown in FIG. 4 eosinophils, a subset of leukocytes (white blood cells), depolarize the right angle of scattered light quantified by the above configuration to a greater degree than other leukocytes. 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 1o regarding leukocytes, and more specifically eosinophils, as shown in FIGS. 6B, 7B, 8B, and 9B, the cluster points within the eosinophil cluster (the cluster points above the angled threshold line on the graphical representation having "DEPOL" as one axis and "ORTHOGONAL" as the other axis) 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 cluster. Additionally, 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.
A need thus exists for a flow cytometer apparatus and related method in which the cell cluster points are less dense for each of characterization of the different cell clusters.
A need also exists for the above apparatus and method which has fewer and less expensive components.
Summary of the Invention According to a first aspect of the present invention there is provided a flow cytometer comprising; 25 a flow cell comprising a passage through which blood cells containing particles to be detected flows; a laser input which emits a beam of light oriented substantially orthogonally to the flow of blood cells through the flow cell such that the laser light impinges upon the particles in the blood cells as they pass through the flow cell; and a right angle scatter light detector being effective to collect a cone of unfiltered right angle scattered light of at least 1000 and convert said unfiltered right angle scattered light into a right angle scattered light signal; and a signal processor, said signal processor being effective to distinguish esoinophils fr-om other leukocytes on the basis of said right angle scattered light signal.
According to a second aspect of the present invention there is provided a method for particle discrimination by light scattering, comprising the steps of: R:\LIBZZ]05880.doc:mrr .IIIII1II1II II.1I 1 1. I lllillY III~Y(I -IYI* I.III-IIIIIIIIII UJ. I~Y IYUI(*(ilYIY*UIIIIIIiil*.iitlllll Ui.l yyilyl U ~l lll. li I~.l~"rrl*r~2rl...flowing a liquid containing biological cells through a flow cell; directing a beam of light in a direction that contacts said biological cells in said flow cell and is substantially orthogonal to a direction of flow of said biological cells through said flow cell; detecting a cone of unfiltered right angle scattered light of at least 1000; converting said detected unfiltered right angle scattered light into a right angle scattered light signal; and identifying eosinophils present among said biological cells on the basis of said right angle scattered light signal.
According to a third aspect of the present invention there is provided a flow cytometer, comprising: a flow cell; a laser input, said laser input emitting a beam of light that is oriented substantially orthogonally to a direction of flow of blood cells through said flow cell; 1i a right angle scatter light detector, said right angle scatter light detector being effective to collect a cone of right angle scattered light of at least 1000 and convert said right angle scattered light into a right angle scattered light signal; and a signal processor, said signal processor being effective to distinguish eosinophils from other leukocytes on the basis of said single right angle scattered light signal.
According to a fourth aspect of the present invention there is provided a method for particle discrimination by light scattering, comprising the steps of: flowing a liquid containing biological cells through a flow cell; S• directing a beam of light in a direction that contacts said biological cells in said flow cell and is substantially orthogonal to a direction of flow of said biological cells through said flow cell; detecting a cone of right angle scattered light of at least 100°; converting said detected right angle scattered light into a single right angle scattered light signal; and S identifying eosinophils present among said biological cells on the basis of said single S 30 right angle scattered light signal.
The high numerical aperture flow cytometer described herein includes a flow cell and a laser input. The laster input emits a beam of light that is oriented substantially orthogonally 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. Unlike the prior art, the laser light (R:\LIBZZ]05880.doc:mrr l Y I. I ~YII^ 3a emitted by the laser input need not be polarised 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 900. This scatter is termed "low angle forward scatter light" and has an angle of from about 20 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 1000 or greater, and preferably 1 30' or greater. It is this larger light cone value over the prior art light cone of about 720 that results in the greater cluster separation in the present invention due to the larger signal gathered. In contrast, the smaller 720 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 50 from the beam of the laser input.
In one embodiment of the present invention, both right angle scatter light detector and low angle forward scatter light detector are employed in order to produce a 2-dimensional **.*cytogram. However, it should be noted that in another embodiment of the 000 **as [R:\LIBZZ]05880doc: mrr 4 present invention, only right angle scatter light detector is employed, low angle forward scatter light detector is not employed, and characterization of eosinophils is possible.
BRIEF DESCRIPTION OF THE DRAWINGS These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
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. 6A is a graphical representation of 2% canine eosinophil data employing the present invention; FIG. 6B is a graphical representation of 2% canine eosinophil data employing the prior art; FIG. 7A is a graphical representation of 8% canine eosinophil data employing the present invention; FIG. 7B is a graphical representation of 8% canine eosinophil data employing the prior art; o• 'UI U' I lll~ UYM W II"IUI rr FIG. 8A is a graphical representation of 10% canine eosinophil data employing the present invention; FIG. 8B is a graphical representation of 10% canine eosinophil data employing the prior art; FIG. 9A is a graphical representation of human eosinophil data employing the present invention; and FIG. 9B is a graphical representation of human eosinophil data employing the prior art.
Detailed Description of the Preferred Embodiments Referring to FIG. 2, 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. Preferably 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 analysis. Laser input 20 emits a beam of light that is oriented substantially orthogonally 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 milliwatts, model No. HL6320G manufactured by Hitachi 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"). A second portion of the beam from laser input 20 that impinges upon the cells in flow cell 18 is scattered at a much lower angle than 900. This scatter is termed "low angle forward scatter light" and has an angle of from about 2' to about 50 from the orientation of the original beam from laser input 20. 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 1000 or greater, and preferably 1 30" or greater. It is this larger light cone value over the prior art light cone of about 720 that results in the greater cluster separation in the present invention due to the larger signal gathered. In contrast, the smaller 720 cone of the prior art results in missed signals and lesser cluster separation.
[R:\L1BZZ]05880.doc:mrT 6 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 50 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.
In one embodiment of the present invention, both right angle scatter light detector 22 and low angle forward scatter light detector 24 are employed in order to produce a 2dimensional cytogram. However, it should be noted that in another embodiment of the present invention, only right angle scatter light detector 22 is employed, low angle 0o forward scatter light detector 24 is not employed, and characterization of eosinophils is possible.
While the electro-optical elements of FIG. 2 have been described above using specific components, it will readily be apparent to one skilled in the art that different components can be employed to achieve the desired results described above. More specifically, reference is made to Practical Flow Cytometry 3 r d Cytometry 3 rd Ed. 1995 by Howard M. Shapiro, Wiley-Liss Publisher, ISBN No. 0-471-30376-3, which is incorporated herein by reference.
Referring to FIG. 3, 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 R
L
[R:kLI BZZ]05880.doc: rrr WO00/49387 PCTIUSOO/04069 7 the voltage or current curve, received from right angle light scatter detector 22 and/or low 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 known methods, including but not limited to those disclosed in Practical Flow Cytometry 3 rd Ed. by Howard M. Shapiro, 1995 Wiley-Liss Publishers, ISBN No. 0-471-30376-3, incorporated herein by reference.
Referring to 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.
6A, 7A, 8A, and 9A, produce cluster points that are less concentrated than are those of the prior art. Computer software programs employed to identify clusters based on cluster points can thus do so more reliably with the present invention.
Next referring to FIGS. 6A, 6B, 7A, 7A, 7B, 8A, 8B, 9A and 9B, graphical representations ofleukocyte identification is shown, with specific reference to eosinophil identification. The data of FIGS. 6A, 7A, 8A, and 9A was employed using the apparatus of the present invention. In FIGS. 6A, 7A, 8A, and 9A the term R2 denotes primarily lymphocytes, R3 denotes primarily monocytes, R4 denotes primarily neutrophils and denotes primarily eosinophils. 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 1 in phosphate buffered saline. Then 40 microliters of the 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 WO 00/49387 PCT/USOO/04069 8 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.
A good correlation exists between the eosinophil of the present invention of FIGS.
6A, 7A, 8A and 9A 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 Regarding FIGS. 7A and 7B, the eosinophil data for the present invention is 7.6% and for the prior art is Regarding FIGS. 8A and 8B the eosinophil data for the present invention is 13.1% and for the prior art is Regarding FIGS. 9A and 9B the eosinophil data for the present invention is 10.8% and for the prior art is 14.6%. For all of the above graphical representations of the present invention, FIGS. 6A, 7A, 8A and 9A an eosinophil cluster is present at R5. Regarding the prior art data of FIGS. 6B, 7B and 8B the SIZE/COMPLEXITY graphical representation shows no eosinophil cluster, while the graphical representation of FIG. 9B does show a cluster.
A comparison of the data of the present invention from FIGS. 6A, 7A, 8A and 9A with the prior art data of FIGS. 6B, 7B, 8B and 9B 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.
While the invention has been described with reference to particular embodiments and applications, it will be appreciated that various embodiments and applications based on the teaching of the present invention are possible.
Claims (23)
1. A flow cytometer comprising; a flow cell comprising a passage through which blood cells containing particles to be detected flows; a laser input which emits a beam of light oriented substantially orthogonally to the flow of blood cells through the flow cell such that the laser light impinges upon the particles in the blood cells as they pass through the flow cell; and a right angle scatter light detector being effective to collect a cone of unfiltered right angle scattered light of at least 1000 and convert said unfiltered right angle scattered light into a right angle scattered light signal; and a signal processor, said signal processor being effective to distinguish esoinophils from other leukocytes on the basis of said right angle scattered light signal.
2. The flow cytometer of claim 1 wherein the laser light which enters the flow cell is not polarised.
3. The flow cytometer of claim 1 or claim 2 wherein the flow cell is quartz.
4. The flow cytometer of any one of claims 1 to 3 wherein the particles are viruses. The flow cytometer of any one of claims 1 to 4, wherein the laser input is a semiconductor diode laser which emits laser light at a wavelength of approximately 635nm. S 20 6. The flow cytometer of any one of claims 1 to 5, wherein the cone of scattered light is at least 130°.
7. The flow cytometer of any one of claims 1 to 6, wherein the light detector is positioned approximately 2mm from the particles in the flow cell. i 8. The flow cytometer of claim 1 wherein another portion of the beam from the laser input which impinges upon the particles as they pass through the flow cell is scattered at an angle of less than 900 from the orientation of the beam from the laser input.
9. The flow cytometer of claim 8, wherein the light scattered at an angle of less than 900 is scattered at an angle of from about 20 to about The flow cytometer of claim 9, wherein a second detector is oriented to receive 30 the beam of light scattered at an angle of from 20 to 50 from the orientation of the beam of the laser input.
11. The flow cytometer of claim 10, wherein the light detector and the second light detector are employed to produce a two dimensional cytogram. [R:\LIBZZ]05880.doc:mrr
12. A high numerical aperture flow cytometer for detecting particles in a fluid sample, substantially as hereinbefore described with reference to any one of Figures 2, 3, 6A, 7A, 8A or 9A.
13. A method for particle discrimination by light scattering, comprising the steps of: flowing a liquid containing biological cells through a flow cell; directing a bean of light in a direction that contacts said biological cells in said flow cell and is substantially orthogonal to a direction of flow of said biological cells through said flow cell; to detecting a cone of unfiltered right angle scattered light of at least 1000; converting said detected unfiltered right angle scattered light into a right angle scattered light signal; and identifying eosinophils present among said biological cells on the basis of said right angle scattered light signal.
14. The method of claim 13, wherein said step of detecting is performed by a right angle scatter light detector located at a distance of about 2 millimeters from said flow cell. The method of claim 13, further comprising collecting low angle forward scattered light at an angle between about 2* to about 5' from said beam of light.
16. The method of claim 13, further comprising detecting a cone of unfiltered right angle scattered light of at least 130'.
17. The method of claim 13, further comprising: detecting said cone of unfiltered right angle scattered light by using a right angle scatter light detector located at adistance of about 2millimeters from said flow cell; *collecting low angle forward scattered light at an agle between about 2"to about from said beam of light; and detecting a cone of unfiltered right angle scattered light of at least 1300.
18. A flow cytometer, comprising: a flow cell; a laser input, said laser input emitting a beam of light that is oriented substantially 30 orthogonally to a direction of flow of blood cells through said flow cell; a right angle scatter light detector, said right angle scatter light detector being effective to collect a cone of right angle scattered light of at least 100" and convert said right angle scattered light into a right angle scattered light signal; and a signal processor, said signal processor being effective to distinguish eosinophils from other leukocytes on the basis of said single right angle scattered light signal. [RzALIBZZ]O588O.doc:p., 11
19. The flow cytometer of claim 18, wherein said right angle scatter light detector is located at a distance of about 2 millimeters from said flow cell. The flow cytometer of claim 18, further comprising at least one low angle forward scatter light detector, said low angle forward scatter light detector being effective to collect low angle forward scattered light at an angle between about 20 to about 5' from said beam.
21. The flow cytometer of claim 18, wherein said right angle scatter light detector is effective to collect a cone of unfiltered right angle scattered light of at least 130'.
22. The flow cytometer of claim 18, further comprising: io a low angle forward scatter light detector, said low angle forward scatter light detector being effective to collect low angle forward scattered light at an angle between about 2" to about 50 from said beam; and a right angle scatter light detector located at a distance of about 2 millimeters from said flow cell and effective to collect a cone of unfiltered right angle scattered light of at least 1300.
23. A method for particle discrimination by light scattering, comprising the steps of: flowing a liquid containing biological cells through a flow cell; directing a beam of light in a direction that contacts said biological cells in said flow cell and is substantially orthogonal to a direction of flow of said biological cells through said flow cell; oooo detecting a cone of right angle scattered light of at least 1000; converting said detected right angle scattered light into a single right angle scattered light :"":signal; and identifying eosinophils present among said biological cells on the basis of said single right angle scattered light signal.
24. The method of claim 23, wherein said step of detecting is performed by a right angle scatter light detector located at a distance of about 2 millimeters from said flow cell.
25. The method of claim 23, further comprising collecting low angle forward S 30 scattered light at an angle between about 2' to about 50 from said beam of light.
26. The method of claim 23, further comprising detecting a cone of right angle scattered light of at least 1300.
27. The method of claim 23, further comprising: detecting said cone of right angle scattered light by using a right angle scatter light detector located at a distance of about 2 millimeters from said flow cell; [R:\LIBZZ]05880.doc: r_ collecting low angle forward scattered light at an angle between about 20 to about from said beam of light; and detecting a cone of right angle scattered light of at least 130°.
28. A method for particle discrimination by light scattering, substantially as hereinbefore described with reference to any one of the Figures 2, 3, 5, 6A, 7A, 8A or 9A.
29. A flow cytometer according to claim 18, substantially as hereinbefore described with reference to any one of the Figures 2, 3, 5, 6A, 7A, 8A or 9A. Dated 17 October, 2003 IDEXX Laboratories, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 9000 o* S s S [RA-LIBZZ]05880.doc:mrT a *ui*mr-?nr* l-u~lvnmt~*YI^Y rur^-~~Na.~~,lllY"Y".II-III YI Un L-U 11.1.1 -1(11 nil.llII.IU.II. UIIIIC- YI -Y-lll. Illiily-.l*i l" rl" 11 111 .UU II
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US12080499P | 1999-02-19 | 1999-02-19 | |
| US60/120804 | 1999-02-19 | ||
| PCT/US2000/004069 WO2000049387A2 (en) | 1999-02-19 | 2000-02-18 | High numerical aperture flow cytometer and method of using same |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| AU3597600A AU3597600A (en) | 2000-09-04 |
| AU768616B2 true AU768616B2 (en) | 2003-12-18 |
| AU768616C AU768616C (en) | 2004-12-16 |
Family
ID=22392650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU35976/00A Ceased AU768616C (en) | 1999-02-19 | 2000-02-18 | High numerical aperture flow cytometer and method of using same |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1095256A2 (en) |
| JP (1) | JP2002537557A (en) |
| AU (1) | AU768616C (en) |
| CA (1) | CA2329031C (en) |
| WO (1) | WO2000049387A2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6149867A (en) | 1997-12-31 | 2000-11-21 | Xy, Inc. | Sheath fluids and collection systems for sex-specific cytometer sorting of sperm |
| US6618143B2 (en) * | 2000-02-18 | 2003-09-09 | Idexx Laboratories, Inc. | High numerical aperture flow cytometer and method of using same |
| AU2002220018A1 (en) | 2000-11-29 | 2002-06-11 | Colorado State University | System for in-vitro fertilization with spermatozoa separated into x-chromosome and y-chromosome bearing populations |
| US7169548B2 (en) | 2002-09-13 | 2007-01-30 | Xy, Inc. | Sperm cell processing and preservation systems |
| EP2306174B1 (en) | 2003-03-28 | 2016-05-11 | Inguran, LLC | Flow cytometry nozzle for orienting particles and corresponding method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5650847A (en) * | 1995-06-14 | 1997-07-22 | Erkki Soini | Method and device for determination of parameters of individual microparticles |
| US5747343A (en) * | 1994-10-31 | 1998-05-05 | Nihon Kohden Corporation | Leukocyte classification reagent |
| US5940177A (en) * | 1997-01-10 | 1999-08-17 | Basf Aktiengesellschaft | Method and apparatus for determining the size distribution of different types of particles in a sample |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4818103A (en) * | 1981-05-15 | 1989-04-04 | Ratcom | Flow cytometry |
| US4606636A (en) * | 1983-10-25 | 1986-08-19 | Universite De Saint-Etienne | Optical apparatus for identifying the individual multiparametric properties of particles or bodies in a continuous flow |
| NL8601000A (en) * | 1986-04-21 | 1987-11-16 | Jan Greve T H Twente Afdeling | THE USE OF POLARIZED LIGHT IN FLOW CYTOMETRY. |
| US5057413A (en) * | 1988-06-13 | 1991-10-15 | Becton, Dickinson And Company | Method for discriminating between intact and damaged cells in a sample |
| US4954715A (en) * | 1989-06-26 | 1990-09-04 | Zoeld Tibor | Method and apparatus for an optimized multiparameter flow-through particle and cell analyzer |
| ATE304170T1 (en) * | 1994-08-01 | 2005-09-15 | Abbott Lab | METHOD AND DEVICE FOR PERFORMING AUTOMATIC ANALYSIS |
| US5631165A (en) * | 1994-08-01 | 1997-05-20 | Abbott Laboratories | Method for performing automated hematology and cytometry analysis |
| JP3504029B2 (en) * | 1995-07-04 | 2004-03-08 | シスメックス株式会社 | Particle analyzer |
| DE59710824D1 (en) * | 1996-05-03 | 2003-11-13 | Ciba Sc Holding Ag | High capacity optical storage media containing xanthene dyes |
| JP3642658B2 (en) * | 1997-06-30 | 2005-04-27 | シスメックス株式会社 | Urine component analyzer and analysis method |
-
2000
- 2000-02-18 EP EP00914609A patent/EP1095256A2/en not_active Ceased
- 2000-02-18 CA CA002329031A patent/CA2329031C/en not_active Expired - Fee Related
- 2000-02-18 JP JP2000600079A patent/JP2002537557A/en active Pending
- 2000-02-18 AU AU35976/00A patent/AU768616C/en not_active Ceased
- 2000-02-18 WO PCT/US2000/004069 patent/WO2000049387A2/en not_active Application Discontinuation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5747343A (en) * | 1994-10-31 | 1998-05-05 | Nihon Kohden Corporation | Leukocyte classification reagent |
| US5650847A (en) * | 1995-06-14 | 1997-07-22 | Erkki Soini | Method and device for determination of parameters of individual microparticles |
| US5940177A (en) * | 1997-01-10 | 1999-08-17 | Basf Aktiengesellschaft | Method and apparatus for determining the size distribution of different types of particles in a sample |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002537557A (en) | 2002-11-05 |
| EP1095256A2 (en) | 2001-05-02 |
| WO2000049387A2 (en) | 2000-08-24 |
| CA2329031A1 (en) | 2000-08-24 |
| AU768616C (en) | 2004-12-16 |
| AU3597600A (en) | 2000-09-04 |
| CA2329031C (en) | 2003-09-23 |
| WO2000049387A3 (en) | 2001-02-15 |
| WO2000049387A9 (en) | 2001-09-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2002327820B2 (en) | High numerical aperture flow cytometer and method of using same | |
| AU2002327820A1 (en) | High numerical aperture flow cytometer and method of using same | |
| US6320656B1 (en) | High numerical aperture flow cytometer and method of using same | |
| EP0442025B1 (en) | Optical particle analyzing apparatus having two types of light sources | |
| EP0698211B1 (en) | Reticulocyte analyzing method and apparatus utilizing light scatter techniques | |
| US4727020A (en) | Method for analysis of subpopulations of blood cells | |
| AU602207B2 (en) | Multi-part differential analyzing apparatus utilizing light scatter techniques | |
| US8767208B2 (en) | System and method for measuring particles in a sample stream of a flow cytometer using low-power laser source | |
| US7274316B2 (en) | System and method for managing data from a flow analyzer | |
| US20040038413A1 (en) | Method and apparatus for differentiating blood cells using back-scatter | |
| US20030048433A1 (en) | Cytometer signal processing system and method | |
| ES8609727A1 (en) | Method and reagent system for four-population differential determination of leukocytes | |
| Leary et al. | Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells. | |
| JPH0129576Y2 (en) | ||
| AU768616B2 (en) | High numerical aperture flow cytometer and method of using same | |
| WO2020147255A1 (en) | Sample optical detection device, sample detection method, and sample analyzer | |
| US5735274A (en) | Apparatus and method for analyzing particles including shifting a judgement region | |
| JP3504030B2 (en) | Method and apparatus for determining particle criterion, and particle analyzer using the criterion | |
| Burger et al. | Acousto‐optic laser‐scanning cytometer | |
| JPS6193932A (en) | Particle analysis instrument | |
| US20140364708A1 (en) | Apparatus for enhancing the mold-in algorithm | |
| JPS62108386A (en) | Particulate counter |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGA | Letters patent sealed or granted (standard patent) | ||
| DA2 | Applications for amendment section 104 |
Free format text: THE NATURE OF THE PROPOSED AMENDMENT IS AS SHOWN IN THE STATEMENT(S) FILED 20040429 |