Abstract:
A method and apparatus for selectively sorting individual particles, such as blood cells of a particular type of interest, from a plurality of particles of different types. Particles of interest are individually differentiated from other particles in a known manner, and a discrete control signal is produced in response to having identified a particle of interest. An impulse generator, acting in response to such control signal, applies a focused impulsive force on the identified particle of interest, such force serving to eject such particle from the plurality of particles of which it is a part. The ejected particles of interested are then collected in a separate container. The apparatus of the invention is preferably embodied in a flow cytometric instrument.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     The present invention relates to improvements in methods and apparatus of the type commonly used to automatically and rapidly sort minute particles, e.g. biological cells, entrained in a moving liquid on the basis of certain predetermined particle characteristics. More particularly, it relates to a particle-sorting method and apparatus in which particles of interest are selectively extracted from their entraining liquid by using a hydraulic impulse to selectively eject such particles of interest from the entraining liquid.  
         [0003]     2. The Related Prior Art  
         [0004]     Flow cytometry is commonly used to differentiate various types of cells and other “formed bodies” comprising a biological fluid, e.g., whole blood. Conventional flow cytometers commonly comprise an optically-transparent flow cell, usually made of quartz, having a central channel through which a stream of cells to be individually identified is made to flow. Movement of the cell stream through the flow cell channel is hydrodynamically entrained to the central longitudinal axis of the flow cell channel by a cell-free sheath liquid that concentrically surrounds the cell stream and flows along with the cell stream as it passes through the flow cell channel. As each cell passes through a cell-interrogation zone of the flow cell channel, it is irradiated with a focused beam of radiation (as commonly provided by a laser source). Upon impinging upon each cell, the laser beam is scattered in a pattern characteristic of the morphology, density, refractive index and size of the cell. Further, the spectral characteristics of the laser beam may act to excite certain fluorochromes associated with selected cells, as may be the case when a cell&#39;s DNA has been previously stained with such fluorochromes, or when a fluorochrome molecule has been previously conjugated with a selected type of cell, either directly or via an intermediate bead or the like. Photodetectors strategically positioned about the optical flow cell serve to convert the light-scattered by each cell and the fluorescence emitted by the excited fluorochromes to electrical signals which, when suitably processed, serve to identify the irradiated cell. In addition to the light scatter and fluorescence measurements made on each cell, some flow cytometers further characterize each cell by measuring certain physical and/or electrical properties of each cell as it passes through the flow cell. Using the well-known Coulter Principle, a DC and/or an RF current is caused to pass through a constricted aperture in the flow cell channel simultaneously with the movement of cells therethrough. The volume of each cell affects the level of DC current through the flow cell aperture, and the cell&#39;s electrical conductivity affects the RF current through such aperture. See, for example, the flow cytometer disclosed in the commonly assigned U.S. Pat. No. 6,228,652, issued in the names of Carlos M. Rodriguez et al.  
         [0005]     A conventional light scatter and fluorescence-sensing flow cytometer of the type noted above is disclosed in U.S. Pat. No. 3,710,933 issued to Mack J. Fulwyler et al. To this standard flow cytometer, Fulwyler et al. have added a cell-sorting component that operates to selectively remove and collect certain cells of interest (e.g., abnormal cells) from the effluent of cells that have already passed through the optical flow cell and have been identified as to cell type. More specifically, the cell-sorting component comprises a piezoelectric device that acts to vibrate the flow cell so as to effect the production a stream of droplets from the cell-entraining sheath liquid exiting from the flow cell. Ideally, each droplet contains but a single cell that has been characterized as to cell type by the light-scatter and fluorescence measurements just made on such cell. Each droplet in the droplet stream is then electrostatically charged as it passes between a pair of electrically charged plates, and each charged droplet is selectively deflected (or not deflected) towards a collection container as it passes between a pair of electrostatically charged deflection plates, such plates being charged to a droplet-deflecting polarity only at a time to deflect droplets (and cells) of interest. The instantaneous polarity of the deflection plates is determined by a cell-characterization processor that processes the cell-measurement signals from the optical flow cell.  
         [0006]     In cell-sorting flow cytometers of the above type, the continuous production of suitably sized droplets can be problematic. Not only is it technically difficult to continuously produce droplets that contain only a single cell, but also the required size of the droplets is so small (aerosol in size) that it is difficult to control their precise movement as they exit from the flow cell. Typically, when it is suspected that a droplet contains more than one cell, the droplet is allowed to proceed to a waste container in order to avoid potential contamination of the collected cells of interest with other cells.  
       SUMMARY OF THE INVENTION  
       [0007]     In view of the foregoing discussion, an object of this invention is to provide an improved particle-sorting apparatus and method that overcomes the above-described technical problems of the prior art technique.  
         [0008]     According to a first aspect of the invention a new and improved method is provided for selectively sorting particles of a particular type of interest from a plurality of particles of different types, including particles of the particular type of interest. Such method generally comprises the steps of: (a) differentiating individual particles of the particular type of interest from other particles in the plurality of particles, and producing a discrete control signal corresponding to the differentiation of each particle of the particular type of interest; (b) selectively producing, in timed relation to the production of each control signal, an impulsive physical force on a specific particle in the plurality of particles whose differentiation has resulted in the production of the corresponding control signal, such impulsive physical force being adapted to eject only such specific particle from the plurality of particles; and (c) collecting the individual particles ejected from the plurality of particles in a separate container. According to a preferred embodiment, the plurality of particles are contained in a liquid medium, and the impulsive physical force is a focused hydraulic force that is applied to a particle of interest within the liquid medium. In a particularly preferred embodiment, the plurality of different particles is entrained as a linear array in a moving stream of liquid. In this case, the particles are characterized as to type, one after another, as they pass a fixed location along the path of the entraining liquid stream. The focused hydraulic force is applied to the entraining liquid at a second location downstream of the fixed location at which each particle is characterized, and in timed relationship to the passage of a particle of interest past such second location. The focused hydraulic force operates to expel a droplet of liquid from the particle-entraining liquid, each droplet so produced containing a particle of interest. Preferably, the method of the invention is carried out in a flow cytometric instrument of the type described herein.  
         [0009]     According to a second aspect of the invention, an improved apparatus is provided for selectively sorting particles of a particular type of interest from a plurality of particles of different types, including particles of the particular type of interest. Such apparatus generally comprises (a) a particle-characterizing component, such as the optical flow cell and its associated particle-detecting components of a conventional flow cytometer, for differentiating individual particles of interest from other types of particles within the plurality of particles, and for producing a control signal in response to having differentiated a particle of interest; (b) an impulse generator operatively coupled to the particle-characterizing component and responsive to a control signal produced thereby to produce a focused physical force adapted to eject a single particle of interest from the plurality of particles of different types; and (c) a container for collecting such ejected particles. Preferably, the impulse generator is a piezo-electrically driven device that operates to provide a focused hydraulic impulse (i.e., a short-lived force) on a moving stream of liquid serving to entrain particles moving along a desired path. Such impulse is timed to eject a liquid droplet from the moving stream of liquid, such droplet containing a particle of interest.  
         [0010]     The particle-sorting method and apparatus of the invention are advantageous over the afore-described electrostatic particle-sorting technique in that there is no need for an electrostatic deflection mechanism and circuitry for electrostatically charging and selectively deflecting particle-containing droplets as a means to sort particles. Thus, the costs of such components are eliminated, as are the attendant disadvantages noted above. In the particle-sorting method of the invention, the only droplets that are formed are those containing the particles of interest. Thus, there is no undesirable aerosol of droplets containing particles of no interest.  
         [0011]     The invention and its advantages will be better understood from the ensuing detailed description of preferred embodiments, reference being made to the accompanying drawings in which like reference characters denote like parts.  
     
    
     BRIEF DESCRIPION OF THE DRAWINGS  
       [0012]      FIG. 1  is a schematic illustration of a prior art system for sorting particles of different types;  
         [0013]      FIG. 2  is a simple timing chart illustrating the time relationship between the detection of a particle and the time at which such particle is sorted by the apparatus of  FIG. 1 ;  
         [0014]      FIG. 3  is an enlarged schematic illustration of a preferred apparatus for sorting particles in accordance with the present invention;  
         [0015]      FIGS. 4 and 5  are perspective and cross-sectional illustrations of preferred apparatus of the invention; and  
         [0016]      FIG. 6  is an enlarged cross-sectional illustration of a portion of the apparatus shown in  FIG. 5 . 
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0017]     Referring now to the drawings,  FIG. 1  illustrates a conventional particle-sorting flow cytometer of the type described above. Briefly, particles to be analyzed and sorted, such as cells of a centrifuged blood sample stored in a container  1 , are injected into a pressurized stream of sheath liquid e.g., saline) provided from a source  12 . The sheath liquid concentrically surrounds the injected particles and causes the particles to travel along the central longitudinal axis A of an optical flow cell  14 . The particles to be analyzed are introduced into the sheath fluid at a rate such that the particles are spaced apart and pass one-at-a-time through the flow cell as a substantially linear array of particles. The flow cell is fabricated from an optically transparent material, typically quartz. As each particle passes through the flow cell, it passes through an interrogation zone Z where it is irradiated by a beam of radiation  16 , commonly provided by one or more continuous-wave lasers  17 . Beam radiation, as modulated by each of the irradiated particles, and/or fluorescent radiation emitted by the irradiated particles, is detected by an array of photodetectors  20 . Some of the individual photodetectors serve to detect light scatter in the forward and side directions, while others serve to detect fluorescence. The respective photodetector outputs  20 A are then processed, in a well-known manner, by a microprocessor-controlled work station  21  to provide a characteristic signature that identifies the type of each particle irradiated. Having identified a particle type, the work station provides a control signal CS to an electrostatic sorting system  24  which operates to sort particles of different types in different containers  30 ,  31 .  
         [0018]     In order to isolate (sort) particles of a particular type from other particles in the sample as they exit the flow cell, a portion of the housing H of the flow cell is vibrated at high frequency by a piezoelectric driver  23  or the like. The effect of such vibration is to cause the stream of sheath liquid exiting the flow cell through a nozzle  18  to break-up into individual droplets  25 , each droplet containing only a single particle P of the sample. The electrostatic sorting system  24  operates in a known manner to selectively deflect, by electrostatic forces, droplets of interest, i.e., those droplets containing particles P′ of interest, into a receptacle  30 , while the majority of droplets are collected in a waste receptacle  31 . The electrostatic sorting system  24  comprises a pair droplet-charging electrodes  26  positioned downstream of the discharge nozzle  18 , on opposite sides of the path of the droplet stream. Electrodes  26  operate under the control of a control circuit  27  and workstation  21  to charge only those droplets containing a particle P′ of interest, as determined by the processed output of the photodetectors  20 . The remaining droplets remain uncharged. Of course, the application of the electrostatic charge to a droplet of interest is timed to coincide with the passage of such droplet past the charging electrodes. A pair of charged deflection plates  28  positioned down-stream of the charging electrodes  26  serves to deflect only the charged droplets  25 ′ into receptacle  30 .  
         [0019]     As noted earlier herein, the electrostatic particle-sorting component of the above-described sorting flow cytometer is problematic in certain respects. Most notably, it requires that every particle to be sorted, whether of interest or not, must be contained in a tiny droplet of a size that can be readily deflected from its normal direction of movement by electrostatic forces. Ideally, only those particles of interest need be confined to such droplets, and the remainder can be propelled directly to waste without any droplet formation. In accordance with the present invention, the electrostatic component of the prior art sorting system has been eliminated in its entirety. In its place, a mechanical particle-sorting mechanism is provided which operates only on the relatively small number of particles of interest (i.e., those particles that are to be separated from the larger mass of other particles in a sample) and allows the remaining particles to proceed along a path without any processing or treatment whatsoever.  
         [0020]     Referring to the schematic illustration of  FIG. 2 , a particle-sorting flow cytometer structured in accordance with a preferred embodiment of the invention comprises an optical flow cell  14  of the general type described above. As in the case of the prior art flow cytometer, particles to be characterized, e.g., blood cells, are provided to an optical flow cell  14  from a source  11 , and a particle-entraining sheath liquid is provided to the flow cell from a suitable source  12 . As entrained by the sheath liquid, the particles pass, one at a time, through a particle interrogation zone Z where they are irradiated by a laser beam  16 , and the scattered radiation and/or fluorescence emanating from the irradiated particle is detected by the photodetector component  20 , described above. The outputs of the individual photodetecting elements are processed by the workstation  21  to identify a particle of interest, and a control signal CS. In accordance with the present invention, such control signal is used to control, via an impulse control circuit  41 , a sorting mechanism  40  that operates to physically eject (as opposed to electrostatically deflect) the individual particles of interest flowing from the flow cell, and, in doing so, to surround such ejected particle with a droplet of sheath liquid. The non-ejected particles remain in the laminar flow of the sheath liquid which is normally directed to waste.  
         [0021]     Referring additionally to the schematic illustration of  FIG. 3 , the above-noted particle-sorting mechanism  40  preferably comprises a flow cell extension element  42  that is affixed, at one end, to the exit end  14 A of the flow cell  14  so as to form a liquid-tight seal therewith. The extension element  42  has a central bore  42 A having a longitudinal axis that coincides with the central axis A of the flow cell. Further, bore  42 A has a size and transverse cross-section selected to match that of the flow cell channel  14 A. The opposite end of the extension element is connected to a tube  44  leading to a waste container  46 . The flow cell extension member  42  further defines a transverse bore hole  42 B that intersects perpendicularly with the central bore  42 A. One end of the transverse bore hole  42  communicates with an impulse generator  50 , described in detail below, and the opposite end of the transverse bore hole is communicates with a container  52  for collecting the particles of interest. On command, the impulse generator  50  operates to produce a transverse, impulsive (i.e., short-lived) force F that is focused on a selected particle P′ positioned at the juncture of bore holes  42 A and  42 B. As a result of this force, a droplet D of sheath liquid containing such particle to be ejected from the flow cell extension element and follow a trajectory T towards the particle-sorting container  52 .  
         [0022]     In accordance with a particularly preferred embodiment, each of the bore holes  42 A and  42 B are circular in transverse cross-section, and each has a diameter of between 150 and 300 microns. Thus, as a result of the pressure pulse applied to the flowing sheath liquid, the size of the droplet surround a particle of interest is of comparable diameter, i.e., between 150 and 300 microns. Preferably, container  52  is supported by a housing  55  connected in an air-tight manner to a lateral side of the extension element  42 . Housing  55  defines a pressure-balancing port  56  in which a small pressure-controlling valve (not shown) is inserted to control the pressure within the container  52  and within the exit side of the transverse bore hole  42 B. Such pressure control, in combination with the surface tension of the flowing sheath liquid, prevents sheath liquid, other than that from which the droplets D are formed, from exiting from the flow channel  42 A through the transverse bore hole  42 B in the absence of a pressure pulse from the impulse generator  50 .  
         [0023]     Referring to  FIGS. 4-6  which illustrate the structural details of a preferred impulse generator  50 , the latter is shown as comprising a metal (e.g., aluminum) housing  60  that defines a conical pressure chamber  62  within. During operation of the apparatus of the invention, chamber  62  becomes filled with sheath liquid which serves as the medium through which a focused hydraulic force is applied to eject particles of interest from the particle stream flowing through the flow cell extension  42 . Preferably, housing  60  is generally conical in shape, as shown in  FIG. 4 ; however, it will be appreciated that it may have any exterior shape whatsoever. At the apex of the conically shaped pressure chamber, the chamber converges towards a small bore hole  64  that communicates with the open end of bore hole  42 B formed in the flow cell extension element  42 . The details of this arrangement are best shown in  FIG. 6 . The enlarged open end  62 B of chamber  62  is sealed by a thin diaphragm  66 , preferably made of stainless steel or brass and having a thickness of about 250 microns (0.010 inch). In the example shown, the open end  62 B of the pressure chamber has a diameter of about 36 mm (1.5 inches); thus, diaphragm  66  must have a somewhat larger diameter, preferably about 62.5 mm (2.5 inches), so as to readily cover the pressure chamber opening. The distance between the diaphragm and the apex  62 A is of the order of 50 mm. (2.0 inches). The diaphragm is held in place atop the end of housing  60  by a circular metal ring  68 . Preferably, ring  68  has an inside diameter that coincides with that of the largest diameter of the conical pressure chamber  62 . Ring  68  is held in place atop diaphragm  66  by a plurality of leg members  70  which extend between a rigid metal cover plate  72  and the outer surface of the ring. A stacked piezoelectric beam  80  is positioned in the space between the inside planar surface  72 A of the cover plate and the center of the diaphragm. The nominal length of beam  80  is about 25 mm. (1.0 inch). In a known manner, the length of the piezoelectric beam can be selectively increased, e.g., by 25 microns, by the application of a suitable driving voltage across the piezoelectrically-active portion of beam  80 . Preferably, the waveform of such driving voltage is in the form of a sawtooth, whereby the beam will rapidly increases in length, followed by a much more gradual return to its nominal length. The relatively sudden increase in beam length causes the nominally planar diaphragm  66  to change shape, becoming convex in the direction from left to right, as viewed in the drawing. The effect of this change in shape is to suddenly reduce the volume of the pressure chamber, thereby producing a focused hydraulic force F (owing to the conical shape of the chamber) on the fluid within the chamber and causing the fluid therein to rapidly exit through the hole  64  formed in the apex of the chamber. The focused hydraulic force so produced will impact that portion of the sheath liquid surround the particle of interest, causing a droplet containing such particle to be ejected from the particle stream and to land in the container  52 .  
         [0024]     From the enlarged view of  FIG. 6 , it will be appreciated that the bores  42 A and  42 B need not be of the same size. As shown, bore hole portion  42 B″ through which the droplets are ejected from the flow cell extension may be made significantly smaller than the bore hole portion  42 B′ through which the particle-ejecting hydraulic force is applied. Similarly, bore hole portion  42 B′ may be significantly smaller that bore hole  42 A through which the particle stream and its entraining sheath liquid pass. For example, bore hole portion  42 B″ may be only 150 microns (0.006 inch) in diameter, while bore hole portion  42 B′ is 300 microns (0.012 inch) in diameter, and bore hole  42 A is 450 microns (0.018 inch) in diameter. The respective bore hole diameters will depend on various system parameters, e.g., the sizes of the particles in the sample, the viscosity of the sheath liquid, etc.  
         [0025]     Using the apparatus of the present invention, particles of interest may be sorted at a rate of up to about 1000 particles per second. While this sorting rate is somewhat slower than that which is attainable by the electrostatic sorting method of the prior art, the apparatus of the invention is significantly less complex, and it avoids the already-noted disadvantages of the prior art technique. Further, the apparatus of the invention operates to displace the particles of interest significantly further from the main particle stream than the prior art technique. For example, 150 micron droplets can be easily projected with a velocity such that the droplets travel 10 cm. horizontally before dropping 2.5 cm. vertically. Such a velocity enables the sorting apparatus to be substantially more compact than convention electrostatic sorting devices.  
         [0026]     The invention has been described with regard to a preferred embodiment. It will be understood, however, that various modifications and changes may be made without departing from the spirit of the invention, and such variations are intended to fall within the scope of the appended claims.