Patent Document

FIELD OF THE INVENTION 
     The present invention relates in general to flow cytometer systems and subsystems thereof, and is particularly directed to a new and improved signal processing and control mechanism therefor, that is operative to monitor a particle sensing zone within a fluid transport chamber for the presence of a particle (e.g., blood cell) traveling therethrough, and to produce an output pulse, whose width is representative of the trajectory and thereby the length of time that the particle is within the particle sensing zone as it travels through the fluid transport chamber. This output pulse is then processed in accordance with geometry parameters of successive time delay zones of the particle fluid transport chamber through which the particle passes, in order to derive a composite time delay between the sensing of the particle to the time at which a fluid droplet containing the particle will break off from the carrier fluid. The composite time delay is employed to accurately establish the time at which the particle is controllably charged as the droplet breaks off from the carrier fluid. 
     BACKGROUND OF THE INVENTION 
     Flow cytometers are instruments that are commonly employed in the medical industry to analyze particles (e.g., blood cells) in a patient&#39;s body fluid as an adjunct to the diagnosis and treatment of disease. As a non-limiting example, during chemotherapy treatment, such instruments may be used to sort and collect healthy blood cells (stem cells) from a quantity of blood that has been removed from a patient&#39;s bone marrow prior to chemotherapy. Once a chemotherapy treatment session is completed, a collected quantity of these cells is the reinjected into the patient, to facilitate migration and healthy blood cell reproduction. 
     In accordance with the customary operation of a flow cytometer, particles to be analyzed, such as blood cells of a centrifuged blood sample are injected from a storage container into a (pressurized) continuous or uninterrupted stream of carrier fluid (e.g., saline) that travels through a carrier fluid transport chamber in which individual particles are sensed and contained within droplets that break off from the fluid stream exiting the fluid transport chamber. As diagrammatically illustrated in  FIG. 1 , which shows a portion of a fluid transport chamber  10  of a ‘SENSE IN QUARTZ’ flow cytometer system, a particle-containing carrier fluid  11  and its surrounding sheath fluid layer  12  are directed along an axial flow direction  13  from a relatively wide diameter portion  14  to a reduced diameter exit orifice  15  of the fluid flow chamber  10 . 
     The particle-carrying fluid  11 , that has been introduced into an upstream zone  21  of the chamber, is intersected at a particle-sensing zone  22  by an output (laser) beam  31  emitted by an optical illumination subsystem, such as one or more lasers  30 . Located optically in the path of the laser output beam  31  after its being intercepted by the carrier fluid stream are one or more sensors of a photodetector subsystem  32 . The photodetecting subsystem is positioned to receive light modulated by the contents of (particles/cells within) the carrier fluid stream, which typically includes light reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell. 
     Downstream of the particle sensing zone  22  is a fluid stream constriction zone  23 , wherein the cross sections of the carrier fluid stream and its surrounding sheath are reduced or constricted, so that the carrier fluid exits the chamber through an exit aperture or orifice  15  at a relatively high velocity relative to its travel within the chamber and enter an air space exit zone  24 . From this location the constricted fluid stream, whose cross section is considerably smaller than during its travel through the fluid transport chamber and is sized to accommodate a single particle, continues on through a droplet separation and charging zone  25 , where a charge is selectively applied to a droplet as the droplet separates or breaks off the fluid stream  11  proper. 
     Conventional SENSE IN QUARTZ technology systems of the type shown in  FIG. 1  incorporate a fixed time delay period between the time a particle is sensed and analyzed in the sensing zone  22  and the time of the application of a droplet sorting charge in the downstream charging and droplet separation zone  25 . The use of a fixed delay constitutes a source of error in the charging/sorting operation due to the fact that not all particles travel at the same effective speed along the transport direction of the fluid, due to the fact that not all particles traverse the same trajectory through the flow chamber. 
     More particularly, established flow inside a chamber can be generally described as parabolic flow to some degree. In parabolic flow within zone  21  there is a relationship between particles flowing along the central axis relative to those particles traveling closer to a wall of the chamber. These particles will continue at their respective (parabola profile based) velocities unless acted upon by an outside influence. Such an influence is created when the fluid flow is forced through a change in geometry, such as the exit orifice  15 . Just upstream of this orifice, there is a constriction of the flow and a subsequent acceleration of the particles relative to the change in cross-sectional area. This acceleration is not uniform and therefore causes a greater acceleration of the particles depending upon wherein the particles are flowing in the fluid stream, thereby separating particles at a faster rate than that occurring in the sensing zone  22  of the chamber. Once the flow exits the area affected by the exit orifice, at zone  24 , the velocity can be assumed to be constant, so that there is no further separation of the particles. 
     This differential in velocity and trajectories may be readily understood by referring to  FIG. 2 , which shows a first particle A traveling along the axis  13  of the carrier fluid channel, and a second particle B that is displaced by some distance from the axis  13  as it travels through the fluid transport chamber. Superimposed on the fluid transport chamber diagram of  FIG. 2  is a set of velocity profiles showing three examples of different velocities associated with different positions of particles relative to the fluid transport axis  13 . In the illustrated example, within the zone  21 , the particle A has a velocity V 1 A which is represented by a velocity vector (arrow  41 ), that coincides with the peak value of a generally parabolic velocity profile of the speed of travel of the carrier fluid through the upstream portion of chamber. 
     Similarly, within the zone  21 , the (off-axis) particle B has a velocity V 1 B, which is represented by a reduced amplitude velocity vector (arrow  42 ), which coincides with a reduced value along the velocity profile of the speed of travel of the carrier fluid through the chamber.  FIG. 2  also shows within the zone  21  a further (off-axis) velocity V 1 C that lies in between associated the velocities V 1 A and V 1 B, and would be associated with an off axis particle (not shown) lying between the coaxial particle A and the off-axis particle B. 
     From  FIG. 2  it is apparent that particles closer to the axis  13  travel at higher velocities as they pass through the sensing zone  22 . The velocity profile overlay of  FIG. 2  also shows that as the particles leave the sensing zone  22  and approach an acceleration zone  23  adjacent to the exit orifice  15 , the speed of the fluid containing the particles must increase in order to comply with the conservation of mass. For the particle A, its velocity increases from a value of V 1 A in the sensing zone  22  along a first acceleration profile f 1A  to the entrance zone  23  to the exit orifice  15 . Thereafter, the velocity of particle A increases (accelerates) slightly along a second acceleration profile f 2A (a) as its passes through the orifice  15  and travels through the freespace region from zone  24  immediately adjacent to the downstream side of orifice  15  to a downstream zone  25  at a final velocity of V 2 A. Likewise, the velocity of particle B velocity increases from a value of V 1 B at the sensing zone along a first acceleration profile f 1B (a), reaching a higher velocity at the entrance zone  23  to the exit orifice  15 . Thereafter, the velocity of particle B increases (accelerates) slightly along a second acceleration profile f 2B (a) as its passes through the orifice  15  and travels through the freespace region from zone  24  immediately adjacent to the downstream side of orifice  15  to a downstream zone  25  reaching a final velocity of V 2 B. 
     From these different velocity profiles it can be seen that the different trajectories of particles A and B cause different arrival times at the point of application of a droplet sorting charge in zone  25 , which is downstream of exit orifice  15 , so that a fixed time delay will not guarantee that the correct particle will be contained in the sorted droplet. In order to maintain conservation of mass, an off-axis particle, such as the particle B, must undergo a more rapid acceleration than the acceleration of particle A, as it approaches zone  23 , so that it will exit the chamber  10  at the speed of the fluid stream. If the two particles A and B are in the sensing zone  22  at the same time, the coaxial particle A, which has the higher velocity, will exit the chamber ahead of the slower particle B, which has to accelerate up to the speed of the fluid stream exiting the chamber at orifice  15 . In an attempt to deal with this problem, conventional systems sort more than one droplet; this, in turn, decreases the purity of the sorted populations and increases the dilution of the particles. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, the drawbacks of using a fixed delay in conventional flow cytometer sorting mechanisms, as described above, are effectively obviated by utilizing the measured velocity of the particle through a sensing zone, together with other analysis parameters, in particular geometry parameters of respectively different zones through the carrier fluid flow chamber, to derive adjusted delays that compensate for the different trajectories of the analyzed particles. As will be described, knowing the relative velocities of the particles allows a calculation to be made of the expected arrival times of particles at the chamber&#39;s output/sorting orifice, and the generation of a set of variable delays to control the time of droplet charge. This variable control of the delay period increases the purity of the sorted sample and reduces its dilution. The speed of travel of the particle can be readily measured using one or more laser beams and determining the time between scatter pulses, as a non-limiting example. In addition, time of flight or pulse width can be used as an additional parameter to interject particle size as a factor in the time delay algorithm. 
     For this purpose, the signal processing system of the present invention comprises a particle sensor such as a photodetector unit, which is operative to generate an output pulse signal that is produced by a particle within the carrier fluid as a result of interaction of that particle and the laser beam illuminating the fluid as it passes through the sensing zone. The pulse signal is coupled to a digital signal processor, which performs a prescribed pulse evaluation routine, including a sort delay calculation, in order to precisely define the time at which a particle-containing drop to be sorted is to be controllably charged by a downstream charging collar, at the point at which the drop of interest breaks off of a stream of droplets that have exited the output orifice of the fluid flow chamber. 
     The width of the pulse signal is indicative of the velocity of the particle within the fluid flow channel, and provides an initial indication of how close to the axis of the fluid stream the particle is traveling. Where the pulse has a prescribed minimum width, it is inferred that the particle has a trajectory that is coincident with the axis of the fluid stream traveling through and exiting the chamber and is moving at the highest velocity possible through the system. In this instance the time delay between the sensing of the particle and the selective sorting thereof at a location downstream of the exit orifice would be a minimum delay. 
     However, trajectories of particles within the carrier fluid can be expected to vary, due to the fact that the cross section of the fluid flow path through the chamber varies as one proceeds in an upstream to downstream direction through the fluid flow chamber from the relatively wide upstream zone where the particles enter the chamber to the constriction at the exit orifice. Thus, an off-axis particle, will have a velocity vector that is less than the peak velocity vector of a particle traveling coincident with the axis through the flow chamber, so that the width of the sensor output pulse produced by an off-axis particle will be wider than that produced by a particle that is traveling on the fluid flow axis. Whether the sensed signal is indicative of a particle to be sorted is determined by a signal classification step, which employs one or more prescribed criteria, such as amplitude, to identify pulses associated with particles as opposed to those associated with noise. Where a particle-representative pulse has been detected, a sort delay calculation step is executed. The pulse width determination information is measured and applied to the sort delay calculation. The output of the sort delay calculation step is a time delay value that will be used to control the time at which a charge is applied to a droplet charging collar thereby charging the droplet containing the particle of interest. 
     In accordance with the present invention, the sort delay calculation is comprised of a plurality of components that are respectively associated with different zones of the flow chamber through which the particle of interest passes. The first zone is the particle sensing zone in which the particle is sensed, as by way of a laser illumination subsystem. The output pulse produced by the particle sensor has rising and falling edges that define the width of the pulse associated with a potential particle of interest. The particle-sensing zone is upstream of two fluid-constricting zones of the fluid flow chamber, so that the particle of interest has some constant velocity in sensing zone. 
     For a particle traveling coincident with the axis of fluid flow, the constant velocity is defined at the peak of the velocity profile through the flow chamber. Within the sort time delay calculation, the first component to be determined is the time its takes the particle to transit the width of the particle sensing zone. 
     A second time delay component is the time delay associated with the travel of the particle from the constant velocity sensing zone through a first acceleration zone leading to the exit orifice. Because there is a constriction of the carrier fluid in this zone, the particle will undergo an acceleration as it approaches the exit orifice. Therefore, the time required for the particle to traverse this distance is equal to the distance divided by the velocity over that distance. Because the particle is undergoing an acceleration in this region the velocity is not constant, and it is necessary to integrate the acceleration function between the zone boundaries at the downstream end of the particle sensing zone and the distance therefrom to the exit orifice. The acceleration function may be determined empirically or deterministically based upon the geometry parameters of the fluid flow system. There is an additional acceleration of the particle through the exit orifice and an associated transit time therefor is calculated. 
     Once the particle has exited the orifice, it is traveling at a constant exit velocity along the axis of the fluid stream. The distance from the exit orifice to the droplet charging location divided by this velocity is the final time component of the composite time delay. In other words, for an arbitrary particle A, a composite time delay T d A may be defined as: 
     
       
         
           
             
               
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                     2 
                   
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     It will thus be appreciated, that from the instant a particle is initially sensed until the particle arrives at the break-off, charging location downstream of the exit orifice, there is a composite delay time T d  equal to the sum of the incremental delay components described. This composite delay time is used to delay the application of a control signal to a droplet charge amplifier the output of which is coupled to an electrostatic charging collar surrounding the travel path of the droplet sequence. The charging collar is positioned vertically downstream of the fluid chamber exit orifice and upstream of an associated set of electrostatic (opposite polarity, high voltage) deflection plates between which the stream of charged droplets pass as they travel downwardly and are either sorted along a sort path into a sorted droplet collection container, or allowed to pass unsorted along a separate travel path into an aborted or discarded waste container. 
     Advantageously, the charging mechanism of the present invention is dynamic, as it adapts to where within the fluid stream in the fluid flow chamber the particles to be sorted are located. This serves to increase the purity of the sorted sample and reduces its dilution. The speed of travel of the particle can be readily measured using one or more laser beams and determining the time between scatter pulses. In addition, time of flight or pulse width can be used as an additional parameter to interject particle size as a factor in the time delay calculation. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  diagrammatically illustrates a portion of a conventional ‘SENSE IN QUARTZ’ flow cytometer system; 
         FIG. 2  shows a set of velocity profiles overlaid on the ‘SENSE IN QUARTZ’ flow cytometer system of  FIG. 1 ; and 
         FIG. 3  diagrammatically illustrates an embodiment of the signal processing system of the present invention and the manner in which it may be readily interfaced with a conventional flow cytometer fluid flow chamber of the type described with reference to  FIGS. 1 and 2 . 
     
    
    
     DETAILED DESCRIPTION 
     Before detailing the architecture and methodology for compensating for variations in particle trajectories in a flowcell cytometer having an electrostatic sorter in accordance with the present invention, it should be observed that the invention resides primarily in a prescribed novel arrangement of conventional analog and digital circuits and components. Consequently, the configurations of such circuits and components and the manner in which they may be interfaced with a cytometer fluid flow chamber and an associated particle droplet charging unit have, for the most part, been shown in the drawings by readily understandable schematic block diagrams, which show only those specific aspects that are pertinent to the present invention, so as not to obscure the disclosure with details which will be readily apparent to those skilled in the art having the benefit of the description herein. Thus, the schematic block diagrams are primarily intended to show the major components of various embodiments of the invention in convenient functional groupings, whereby the present invention may be more readily understood. 
     Attention is now directed to  FIG. 3 , wherein an embodiment of the signal processing system of the present invention and the manner in which it is interfaced with a conventional flow cytometer fluid flow chamber of the type described with reference to  FIGS. 1 and 2  is diagrammatically illustrated. As shown therein, the system comprises a sensor  201 , such as a photodetector unit, which is operative to generate an output pulse signal  202  that is produced by a particle  203  within the carrier fluid  204  as a result of interaction of that particle and the laser beam illuminating the sensing zone  22 . The pulse is coupled to a digital signal processor  200 , which performs a prescribed pulse evaluation routine, including a sort delay calculation, in order to precisely define the time at which a particle-containing drop to be sorted is to be controllably charged by a downstream charging collar, at the point at which the drop of interest breaks off of a stream of droplets that have exited the output orifice  206  of the fluid flow chamber. 
     The width of the pulse signal  202  is indicative of the velocity of the particle within the fluid flow channel, and provides an initial indication of how close to the axis of the fluid stream the particle is traveling. Where the pulse has a prescribed minimum width, which can be determined experimentally or deterministically, it is inferred that the particle has a trajectory that is coincident with the axis of the fluid stream traveling through and exiting the chamber (which would correspond to the particle A, described above), and is therefore moving at the highest velocity possible through the system. In this instance the time delay T d A between the sensing of the particle and the selective sorting thereof at a location  205  downstream of the exit orifice  206  would be a minimum delay. 
     As described above, however, the trajectories of particles contained within the carrier fluid can be expected to vary, due to the fact that the cross section of the fluid flow path through the chamber  10  varies as one proceeds in an upstream to downstream direction through the fluid flow chamber from the relatively wide upstream zone  21  where the particles enter the chamber to the constriction at the exit orifice  15 . As pointed out above, and as shown in  FIG. 2 , an off-axis particle, such as particle B, has a velocity vector ( 42 ) which is less than the peak velocity vector  41  of a particle A along the axis through the flow chamber  13 , so that the width of the sensor output pulse produced by particle B will be wider than that produced by particle A. Whether the sensed signal is indicative of a particle to be sorted is determined by a signal classification step  221 . If the answer to this step is YES as shown at  222 , a sort delay calculation step  212  is executed, as will be described. The pulse width determination information is measured at  211  and applied to the sort delay calculation  212 . The output of the sort delay calculation step is a time delay value that will be used to control the time at which a charge is applied to a droplet charging collar thereby charging the droplet containing the particle of interest. 
     In accordance with the present invention, the sort delay calculation is comprised of a plurality of components that are respectively associated with different zones of the flow chamber through which the particle of interest passes. The first zone is the sensing zone  22  at which the particle is sensed by the laser illumination subsystem  30 . The output pulse of the sensor  201  produces rising and falling edges that define the width of the pulse associated with a potential particle of interest. Sensing zone  22  is upstream of the two fluid-constricting zones  23  and  24  of the fluid flow chamber, so that the particle of interest has some constant velocity at zone  22 . For the particle A, the constant velocity is V 1 A (represented by arrow  42  in  FIG. 1 ) and is defined at the peak of the (parabolic) velocity profile  40 . Within the sort time delay calculation, the first component to be determined is the time its takes the particle to transit the distance D 2 , which corresponds to the distance (D) of travel through the sensing zone  22 . This delay component for particle A is denoted as time delay T d2 A. 
     A second time delay component is the time delay associated with the travel of the particle from the sensing zone  22  to a first acceleration zone  23 . The associated distance is denoted as distance D 3 . Because there is a constriction of the carrier fluid in this region, the particle A will undergo an acceleration f 1A (a) as it approaches the exit orifice  15 . Therefore, the time T d23 A required for the particle A to traverse the distance D 3  is equal to the distance D 3  divided by the velocity over that distance. Because the particle A is undergoing an acceleration in this region the velocity is not constant, and it is necessary to integrate the acceleration function f 1A (a) between the zone boundaries at the downstream end of distance D 2  and the distance D 3 . The acceleration function f 1A (a) may be determined empirically or deterministically based upon the geometry parameters of the fluid flow system. 
     As a non-limiting example, where the acceleration zone is a uniform symmetric reduction of the cross-sectional area of the flow, the acceleration function would be a constant (k) that can be determined empirically or using numerical fluid flow simulation calculations. In this case:
 
f 1A =k
 
     In general, the time T d23 A required for the particle A to traverse the distance between zones  22  and  23  is definable as: 
     
       
         
           
             
               
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     Similarly, the time T d34 A required for the particle A to traverse the length of the orifice between zones  23  and  24  is definable as: 
     
       
         
           
             
               
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     As a non-limiting example, where the acceleration zone is a uniform symmetric reduction of the cross-sectional area of the flow, the acceleration function would be a constant (k) that can be determined empirically or using numerical fluid flow simulation calculations. In this case:
 
f 2A =k
 
     Once the particle has exited the orifice  15 , it is traveling at a constant exit velocity V 2 A along the axis of the fluid stream. The distance D 5  from the exit orifice to the droplet charging location divided by this velocity is the time T 45 A required for the particle to travel the distance D 5 . 
     It can be seen, therefore, from the instant that the particle A was initially sensed by sensor  201  until the particle arrives at location  205  downstream of the exit orifice  206 , a composite delay time T d A equal to the sum of the incremental delay components described above has elapsed. This composite delay time is used to delay the application of a control signal to a droplet charge amplifier  207 , the output of which is coupled to an electrostatic charging collar  208  surrounding the travel path of the droplet sequence. Charging collar  208  may comprise a metallic cylinder that is located so as to surround the location along the droplet sequence travel path where the individual droplets separate from the fluid stream, and is typically several droplets in length. The charging collar is positioned vertically downstream of the fluid chamber exit orifice  206  and upstream of an associated set of electrostatic (opposite polarity, high voltage) deflection plates  209  and  210 , between which the stream of charged droplets pass as they travel downwardly and are either sorted along a sort path  213  into a sorted droplet collection container  214 , or allowed to pass unsorted along a separate travel path into an aborted or discarded waste container  215 . 
     In other words, for particle A, a composite time delay T d A may be defined as: 
     
       
         
           
             
               
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                 d 
               
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     Likewise, for the case of particle B, which has an initially lower velocity than particle A, from the instant that the particle B is sensed by sensor  201  until particle B arrives at location  205  downstream of the exit orifice  206 , a composite delay time T d B equal to the sum of the incremental delay components described above has elapsed. For particle B, a composite time delay T d B may be defined as: 
     
       
         
           
             
               
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             = 
             
               
                 
                   
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     From the velocity profiles of  FIG. 2 , it can be seen that particle A is accelerated to its maximum velocity through the output orifice well in advance of the time it takes for particle B to be accelerated to its maximum velocity through the output orifice. Consequently, the composite delay for particle A from the time it is sensed in the sensing zone  22  until it is charged by the charging collar  208  will be shorter than the composite delay for particle B. It will be appreciated, therefore, that the charging mechanism of the present invention is dynamic, as it adapts to where within the fluid stream in the fluid flow chamber the particles to be sorted are located. As pointed out above, this serves to increase the purity of the sorted sample and reduces its dilution. The speed of travel of the particle can be readily measured using one or more laser beams and determining the time between scatter pulses. In addition, time of flight or pulse width can be used as an additional parameter to interject particle size as a factor in the time delay calculation. 
     While I have shown and described an embodiment in accordance with the present invention, it is to be understood that the same is not limited thereto but is susceptible to numerous changes and modifications as known to a person skilled in the art, and I therefore do not wish to be limited to the details shown and described herein, but intend to cover all such changes and modifications as are obvious to one of ordinary skill in the art.

Technology Category: 3