EP1242804A2 - Reactif d'enveloppe a viscosite elevee pour cytometrie de flux - Google Patents

Reactif d'enveloppe a viscosite elevee pour cytometrie de flux

Info

Publication number
EP1242804A2
EP1242804A2 EP00989570A EP00989570A EP1242804A2 EP 1242804 A2 EP1242804 A2 EP 1242804A2 EP 00989570 A EP00989570 A EP 00989570A EP 00989570 A EP00989570 A EP 00989570A EP 1242804 A2 EP1242804 A2 EP 1242804A2
Authority
EP
European Patent Office
Prior art keywords
fluid
flow
viscosity
sheath
increasing agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00989570A
Other languages
German (de)
English (en)
Inventor
Richard Channing Moore
Anthony Ferrante
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Union Biometrica Inc
Original Assignee
Union Biometrica Inc
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 Union Biometrica Inc filed Critical Union Biometrica Inc
Publication of EP1242804A2 publication Critical patent/EP1242804A2/fr
Withdrawn legal-status Critical Current

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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
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • 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
    • 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
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • G01N15/1409—Handling samples, e.g. injecting samples
    • G01N2015/1411—Features of sheath fluids

Definitions

  • This invention pertains to a technology for counting and analysis of particles, generally called flow cytometry. Specifically, the invention deals with improvements which allow flow cytometry devices to: 1) handle large particles, including small elongate multicellular organisms, which are considerably larger than the particles on which flow cytometry machines are usually used, and/or 2) operate at significantly increased speeds.
  • Flow cytometry instruments optically analyze particles suspended in a fluid stream as the particles pass through a focussed light beam.
  • the instruments use hydrodynamic focusing to center particles in the fluid stream.
  • the fluid flow must be fully laminar, with no oscillations or turbulence. Needless to say, any imperfections in the hydrodynamic focusing degrade the performance of the instrument. Imperfect hydrodynamic focusing results in flow instabilities, and optical measurements are predicated upon the analyzed particle passing at a constant velocity through the center of the optical beam. In the face of instabilities and imperfect focussing these assumptions are not met and the resulting optical data are erroneous.
  • a sheath stream and sample fluid stream (containing suspended particles to be analyzed) are introduced into the flowcell in a pre-analysis section (chamber) of the flowcell.
  • the sheath stream is injected into the flowcell and allowed to flow for a sufficient distance to form a fully developed laminar flow profile.
  • the sample stream is injected into the center of this flow profile.
  • the sample fluid is thus kept centered in the flow channel of the flowcell by the laminar sheath flow that includes a velocity differential between the sheath and the sample streams.
  • the centered sample particles are analyzed as they pass through an "interrogation" station where a beam such as a laser beam traverses the flowcell and strikes the suspended particles one by one. Light emitted or scattered by the particles is received by one or more optical detectors that output optical data in response to the incident light.
  • the fluid stream is aimed into the well of a microtiter plate or other suitable receptacle.
  • the entire fluid stream would be deposited in the well were it not for the "fluid switch".
  • the fluid switch consists of a separate stream of focussed fluid which strikes the stream in air just below the flow cell.
  • the separate stream diverts the sample stream in air so that it goes to waste and does not enter the microtiter well. Because this intersection occurs below the flowcell, any shock waves or instabilities caused by the intersection are not transmitted upstream into the analysis region where they would spoil the analysis.
  • the "fluid switch" diverting fluid stream (control stream) is controlled by a high-speed fluidic valve.
  • the high-speed valve is closed at the correct instant to cause a section of stream in air containing the organism to pass unimpeded into the microtiter well. Then, the high-speed valve is reopened once again to divert the sample stream in air and prevent additional fluid or organisms from entering the microtiter well.
  • the microtiter tray is moved mechanically to bring another well into position to receive the stream in air, and the entire process is repeated to deposit a single desired organism in the well. In this way each of the wells of the microtiter tray receive a single selected organism.
  • FIG. 1 represents a simplified block diagram of such an instrument.
  • a source of suspended multicellular sample organisms 20 flow into the flowcell 24 which is represented by a dotted line. Sheath fluid from a container 22 enters the flowcell 24 and laminar flow develops as discussed below.
  • a laser beam (not shown) traverses the flow cell 24 and illuminates the organisms. Emitted and scattered light are received by optical detectors 28.
  • the signals are analyzed by a computer 30.
  • the computer output controls a fluidic valve 34 which switches fluid (i.e., compressed gas) from a source 36.
  • a control stream 38 emerging from the valve 34 is aimed onto a sample stream in air 40 that emerges from the lower end of the flowcell 26 forming a deflected stream 42 which goes to waste.
  • the deflected stream may be a mist of droplets.
  • Becton Dickinson and Company manufacture instruments, including the FACStar Plus and the FACScaliber, which are available with special flowcells with larger than normal flow channels. These instruments are intended for use with samples suspended in water, buffer or biological saline.
  • the present inventors have found significant advantages to the use in flow-cytometry instruments of a sheath fluid with a viscosity significantly higher than that of water or biological saline. There are two primary situations where a viscous sheath is of especial advantage:
  • the present inventors have found that increasing the viscosity of the sheath fluid dramatically decreases the flow length required to stabilize fully the flow.
  • This invention is critical for systems which require that the stream exit into air as a solid stream and not as droplets. If the flow rate is too slow, the stream will form droplets and drip out of the exit nozzle.
  • the high-viscosity sheath allows these systems to be run at a flow rate sufficiently high that the fluid exits the nozzle as a solid stream rather than as a series of drops.
  • An additional advantage of the high-viscosity sheath fluid is that large particles often settle out of the sample being analyzed before it reaches the flowcell. Increased viscosity of the sample fluid slows the rate at which the particles settle, making mixing of the samples easier and preventing settling in the sample lines.
  • sheath and sample fluid must have the same refractive index or the sample fluid will scatter light even when no particle is present. This means that modification of sheath viscosity will normally require a similar modification of the sample fluid.
  • the preferred embodiment of this invention consists of a short pre- analysis chamber, in which the sheath is delivered aligned with the axis of the flowcell (for example, through two opposed ports), an analysis chamber with a wide (1 mm) flow channel, and a nozzle through which the flow stream is discharged to air at the downstream end of the analysis section.
  • a solid stream of fluid is diverted by a switchable fluid stream (e.g. gas), which is turned off to dispense a particle.
  • a switchable fluid stream e.g. gas
  • the preferred agent is polyvinyl pyrollidone (PVP). This material can be effectively used over a considerable range of solution concentrations and molecular weight compositions.
  • a 5% by weight solution of a polymer with a 40,000 average molecular weight is effective.
  • Increasing the molecular weight of the polymer generally increases viscosity so that lower concentrations can be used.
  • An ideal solution is a 0.9% by weight solution of a polymer with an average molecular weight of 1.3 million.
  • FIGURE. 1 shows a block diagram of a sorting flow cytometer of the type described in the present invention.
  • FIGURE. 2 is a sectional view of a simple flow cell with the boundary layers shown to describe the development of laminar flow.
  • FIGURE 3 is an enlargement of detail '3' from FIG. 2, showing the pre-analysis section of the flowcell near the sheath inlet.
  • FIGURE 4 is an enlargement of detail '4' from FIG. 2, showing the flowcell exit with a droplet forming.
  • FIGURE 5 is a detail from FIG. 4 and shows a control volume for the droplet.
  • sheath and sample fluids are introduced into a flowcell in a pre-analysis section.
  • the sheath fluid forms a fully developed laminar flow profile within a short flow length (the entry length), and the sample fluid is injected into the center of this flow profile.
  • the sample fluid, containing a single file sequence of particles to be analyzed is kept centered in the flow channel by the laminar sheath flow, and is analyzed as it passes through an "interrogation" station (sensing zone) such as a laser beam traversing the flowcell, combined with one or more optical detectors.
  • the laser beam strikes the sample particles one by one as they pass through the interrogation station at a constant velocity.
  • Light scattered and emitted by each particle is detected by a series of optical detectors whose outputs are data that describe the optical characteristics of the analyzed particle. Because the sample particles are all centered, each particle shows a similar optical interaction with the laser beam. In the case of elongate multicellular organisms the centering process also aligns the long axis of the organism with the direction of flow. If the particles move from side to side as they pass through the laser beam, the detector data would be spurious due to fluctuations introduced by such random movement.
  • the flow passes into the analysis chamber or region of the flowcell, where the particles are measured and analyzed.
  • the rate at which the particles pass through the flowcell depends on the velocity of the fluid flow, which in turn depends on the flow rate.
  • flow rate cannot always be increased to increase the rate of particle analysis because above a certain velocity, the flow in the analysis chamber becomes unstable, laminar flow is lost and accurate measurements can no longer be made.
  • the present inventors have shown that increasing the viscosity of the sheath fluid increases the velocity at which the transition to unsteady flow occurs.
  • FIG. 2 shows a diagrammatic representation of a flowcell similar to one described by Shapiro (Shapiro Howard M. "Practical Flow Cytometry _ _
  • Sheath fluid enters through a sheath inlet tube 1 and enters the pre-analysis section of the cell through an orifice 11.
  • Sample fluid enters through a sample injector tube 2 and is injected into the center of the sheath flow through a second orifice 6.
  • the sheath fluid develops boundary layers 12 extending from the inside chamber wall and the outside wall of the sample injector tube 2.
  • the boundary layers converge at a point 5.
  • the flow converges and flows through an analysis section 8 of the cell, shown here as a quartz capillary.
  • the fluid exits the cell at a nozzle tip 9 and may form into a droplet 10 as shown or a solid stream in air (not illustrated) depending on the flow rates as discussed below.
  • FIG. 3 shows a close-up view of the boundary layers 17 to illustrate the development of laminar flow as discussed below.
  • the boundary layers are areas of viscous flow growing from the walls of the cavity, surrounding an area of inviscid flow 14 at the center.
  • the overall velocity profile is shown at an arbitrary point as 13.
  • Brodskey (Brodskey Robert S. "The Phenomena of Fluid Motions", 1995 Dover Publications Inc. Mineola NY., p. 120) gives an equation for a boundary layer growing on a flat plate as having the form:
  • boundary layers form simultaneously on both the inner and outer radii of an annular cavity, although the coefficient K will be different for the inner and outer layers, with the inner layer developing less quickly since the wall surface is smaller there.
  • the centerline velocity will be determined by the flow rate and geometry; generally for a cylindrical chamber:
  • FIG. 4 shows a small droplet forming on the exit nozzle 27, along with the velocity profile inside the nozzle.
  • FIG. 5 shows a control volume consisting of the droplet, with one entrance plane cutting across the tip of the outlet nozzle 27.
  • UCL is the centerline velocity and R is the radius of the tube. This will be the profile 29 as the fluid crosses the entrance plane of the droplet.
  • the momentum flux term can be found by integrating the differential momentum across the capillary:
  • the rate of flow required to sustain a "solid" stream of fluid in air is directly related to the diameter of the flow channel. Recall that such a solid stream is a prerequisite for the fluid switch sorting arrangement.
  • a flow rate of approximately 2.5 mL/min is needed to ensure a solid stream.
  • the required flow rate goes up eight fold to approximately 20 mL/min.
  • the flow rate increases to approximately 50 mL/min, a twenty-fold increase.
  • the maximum laminar flow rate is 14 mL/min, but the minimum rate for a solid exit stream is 20 mL/min.
  • PVP polyvinyl styrene
  • the maximum stable flow rate increases to over 40 mL/min while the minimum rate for a solid exit stream increases to 29 mL/min. That is, increased viscosity results in a higher laminar flow rate.
  • viscosity altering agents often increase the fluid surface tension, which results in an increase in the minimum flow rate necessary to sustain a solid flow stream. Because the viscosity effect is linearly related whereas the surface tension effect is related as the square root, a given viscosity-altering agent may actually render a given flowcell design useable where the same design would not operate at all with water.
  • Increased viscosity can be achieved with any number of additives dissolved in water, including polyvinyl pyrollidone (PVP), polyethylene glycol (PEG), polyvinyl alcohols, polyvinyl acetals, polyacrylic acids, polyacrylamides, plant gums (such as gum acacia and gum traganth), cellulose ethers (carboxymethyl cellulose), celluloses, hemicelluloses, dextrans, inulins, sucrose and other carbohydrates (monosaccharides, oligosaccharides and polysaccharides).
  • Non-aqueous fluids glycerol, propylene glycol, etc.
  • biological objects require a medium that is at least partially aqueous. For analysis of non-biological objects the fluids can be completely non-aqueous.
  • This invention is an improvement over current flow cytometry methods because it allows the flow cell channel to be enlarged without impairing other functions of the instrument.
  • use of a larger channel requires a decrease in the velocity at which the samples pass through the flowcell to ensure laminar flow, thereby limiting speed.
  • a larger channel also requires that the pre- analysis chamber or region be elongated to allow a sufficient distance for the sheath fluid to develop a steady laminar flow.
  • the current invention allows the use of a larger flow channel without any increase in the length of the pre-analysis chamber or decrease in fluid velocity.
  • the high-viscosity sheath reagent can be used to increase the analysis rate in a standard flow cytometer. Instead of running particles through a larger flow channel at the same velocity, particles can be run through the same flow channel at a higher velocity.
  • This invention is especially useful for systems which require that the stream exit into air as a solid stream and not as droplets. If the flow rate is too slow, the stream will form droplets and dribble from the exit nozzle. However, when the flow rate is increased to ensure formation of a solid stream, laminar flow in the flowcell may be lost unless the entry length is increased. As explained above, this entire problem is greatly exacerbated when the flow channel diameter is increased to accommodate elongated multicellular organisms. The use of high- viscosity sheath allows these systems to be run at a sufficiently high flow rate that the fluid exits the nozzle as a solid stream rather than as drops without impairing laminar flow.
  • sheath fluid One further advantage of the high-viscosity sheath fluid is that large particles often settle out of the sample fluid being analyzed before it reaches the flowcell. Increased viscosity of the sample fluid slows the rate at which the particles settle, making mixing of the samples easier and preventing settling in the sample lines.
  • the sheath and sample fluid must have the same refractive index or the sample fluid will scatter light even when no particle is present. This means that modification of sheath viscosity will normally require a similar modification of the sample fluid so that the indices of refraction match.
  • sucrose, glycerin and other low molecular weight compounds can be employed for viscosity modification, such materials will often have a significant osmotic effect at concentrations sufficient to significantly alter the viscosity.
  • excess osmoticum can distort the samples and even lead to a loss of viability. Therefore, it is preferred to use agents with a higher molecular weight, such as PEG polymers, PVP polymers or carbohydrate polymers. With such agents a significant increase in viscosity can be achieved with only a negligible increase in osmotic strength.
  • a potential drawback to the viscosity-increasing agents is that they generally increase the surface tension of the fluid. This requires a higher flow rate to ensure formation of a solid stream in air.
  • the surface tension effect is related to the square root of the surface tension increase while the velocity change in achieving laminar flow is linearly related to the increase in viscosity, with many agents the improvement due to viscosity increase more than outweighs the problems caused by an increase in surface tension.
  • a viscosity-increasing agent one should select agents that cause the largest increase in viscosity on a mole per mole basis while causing the smallest increase in surface tension on a molar basis.
  • a preferred sheath and sample fluid contain about 0.9% by weight PVP having a molecular weight of about 1.3 million.
  • PVP having a molecular weight of about 1.3 million.
  • testing of potential new drug compounds is a preferred use of the current invention. Therefore, long- term viability of the analyzed organisms is key.
  • the present inventors have tested the viability of Drosophila melanogaster larvae in both 5% PVP (40,000 MW) and 0.9% PVP (1.3 million MW) as well as a variety of concentrations and molecular weights between these figures and have found little, if any toxicity. This is hardly surprising since PVP use is allowed in a large number of food and medical products ranging from beer to hair preparations to eye drops.
  • PVP has been even used as a substitute for human plasma.
  • the overall viability exceeded 95% even after aerating the embryos for 8 hours in PVP with an antifoaming agent.
  • viability in PVP still exceeded 85%

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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)

Abstract

L'invention concerne un cytomètre de flux destiné à l'analyse de particules de grande taille ou de petits organismes multicellulaires. Ce cytomètre risque d'être perturbé par un flux instable étant donné le diamètre important de la chambre d'écoulement. L'utilisation d'un fluide d'enveloppe ou d'une enveloppe et d'un échantillon, outre l'ajout d'un agent renforçateur de viscosité conférant une viscosité supérieure à celle de l'eau, constitue une garantie que le flux dans la section de pré-analyse sera un flux laminaire entièrement développé et que le flux dans la section d'analyse sera laminaire. Cela permet d'effectuer une analyse et un classement précis de particules de grande taille ou une analyse et un classement précis de particules plus petites à une vitesse accrue. L'utilisation de polymères solubles dans l'eau est recommandée du fait qu'ils augmentent la vitesse du fluide et ne produisent presque aucun effet osmotique. Une solution de 0,9 % en poids de pyrollidone polyvinylique présentant un poids moléculaire moyen de 1,3 million est particulièrement efficace. L'utilisation d'agents renforçateurs de viscosité entraînant une augmentation minimale de tension superficielle du fluide est également recommandée.
EP00989570A 1999-12-29 2000-12-29 Reactif d'enveloppe a viscosite elevee pour cytometrie de flux Withdrawn EP1242804A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US17357299P 1999-12-29 1999-12-29
US173572P 1999-12-29
PCT/US2000/035543 WO2001048455A2 (fr) 1999-12-29 2000-12-29 Reactif d'enveloppe a viscosite elevee pour cytometrie de flux

Publications (1)

Publication Number Publication Date
EP1242804A2 true EP1242804A2 (fr) 2002-09-25

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Country Status (5)

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EP (1) EP1242804A2 (fr)
JP (1) JP2004500562A (fr)
AU (1) AU2606301A (fr)
CA (1) CA2396015A1 (fr)
WO (1) WO2001048455A2 (fr)

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US7116407B2 (en) 1998-12-15 2006-10-03 Union Biometrica, Inc. System for axial pattern analysis of multicellular organisms
US7468789B2 (en) * 2004-02-05 2008-12-23 Advanced Analytical Technologies, Inc. Flow cytometer for rapid bacteria detection
JP4606261B2 (ja) * 2005-07-13 2011-01-05 三井造船株式会社 粒子測定方法
US7867778B2 (en) * 2007-02-23 2011-01-11 Visiongate, Inc. Fluid focusing for positional control of a specimen for 3-D imaging
CN103543093B (zh) * 2007-06-07 2016-08-10 技术研究及发展基金有限公司 细胞测量设备
JP2009115672A (ja) * 2007-11-08 2009-05-28 Sony Corp 微小粒子の光学的測定方法及び分取方法、並びに前記光学的測定方法及び分取方法に用いる流路、光学的測定装置及びフローサイトメータ
JP4556996B2 (ja) * 2007-12-13 2010-10-06 ソニー株式会社 光学的検出方法
EP3950136A1 (fr) 2011-03-09 2022-02-09 Pixcell Medical Technologies Ltd. Cartouche jetable de préparation d'un fluide-échantillon contenant des cellules à analyser
KR102053487B1 (ko) 2013-03-15 2019-12-06 아이리스 인터내셔널 인크. 혈액 샘플에서의 입자 분석을 위한 시스 유체 시스템 및 방법
US9857361B2 (en) * 2013-03-15 2018-01-02 Iris International, Inc. Flowcell, sheath fluid, and autofocus systems and methods for particle analysis in urine samples
US10705008B2 (en) 2013-03-15 2020-07-07 Iris International, Inc. Autofocus systems and methods for particle analysis in blood samples
WO2017167361A1 (fr) * 2016-03-30 2017-10-05 Siemens Healthcare Gmbh Alignement d'une entité biologique non sphérique dans un flux d'échantillon à l'aide de flux de fluide viscoélastique ambiant
JP6716057B2 (ja) * 2016-04-08 2020-07-01 富山県 細胞を分離する方法及び装置
SG11202002014PA (en) * 2017-09-19 2020-04-29 Hifibio Sas Particle sorting in a microfluidic system
EP4124846A1 (fr) * 2021-07-29 2023-02-01 Technische Universität München Détection d'agrégats cellulaires en utilisant la microscopie quantitative à contraste de phase
WO2023006372A1 (fr) * 2021-07-29 2023-02-02 Technische Universität München Détection d'objets biologiques moléculaires, d'objets biologiques cellulaires et d'agrégats cellulaires par microscopie quantitative à contraste de phase

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Also Published As

Publication number Publication date
CA2396015A1 (fr) 2001-07-05
JP2004500562A (ja) 2004-01-08
WO2001048455A2 (fr) 2001-07-05
AU2606301A (en) 2001-07-09
WO2001048455A3 (fr) 2002-05-10

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