Patent Publication Number: US-2007102374-A1

Title: Blood processing apparatus with controlled cell capture chamber and method background of the invention

Description:
This application is related to U.S. Pat. No. 5,722,926, issued Mar. 3, 1998; U.S. Pat. No. 5,951,877, issued Sep. 14, 1999; U.S. patent 6,053,856, issued Apr. 25, 2000; U.S. patent 6,334,842, issued Jan. 1, 2002; U.S. patent application Ser. No. 10/884,877 filed Jul. 1, 2004; and U.S. patent application Ser. No. 10/905,353, filed Dec. 29, 2004. The entire disclosure of each of these U.S. patents and patent applications is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION  
      The present invention relates to an apparatus and method for separating particles or components of a fluid. The invention has particular advantages in connection with separating blood components, such as white blood cells and platelets.  
     DESCRIPTION OF THE RELATED ART  
      In many different fields, liquids carrying particle substances must be filtered or processed to obtain either a purified liquid or purified particle end product. In its broadest sense, a filter is any device capable of removing or separating particles from a substance. Thus, the term “filter” as used herein is not limited to a porous media material but includes many different types of devices and processes where particles are either separated from one another or from liquid.  
      In the medical field, it is often necessary to filter blood. Whole blood consists of various liquid components and particle components. The liquid portion of blood is largely made up of plasma, and the particle components include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). While these constituents have similar densities, their average density relationship, in order of decreasing density, is as follows: red blood cells, white blood cells, platelets, and plasma. In addition, the particle components are related according to size, in order of decreasing size, as follows: white blood cells, red blood cells, and platelets. Most current purification devices rely on density and size differences or surface chemistry characteristics to separate and/or filter the blood components.  
      Typically, donated platelets are separated or harvested from other blood components using a centrifuge. White cells or other selected components may also be harvested. The centrifuge rotates a blood separation vessel to separate components within the vessel or reservoir using centrifugal force. In use, blood enters the separation vessel while it is rotating at a very rapid speed and centrifugal force stratifies the blood components, so that particular components may be separately removed. Components are removed through ports arranged within stratified layers of blood components.  
      White blood cells and platelets in plasma form a medium density stratified layer or “buffy coat”. Because typical centrifuge collection processes are unable to consistently and satisfactorily separate white blood cells from platelets in the buffy coat, other processes have been added to improve results. In one procedure, after centrifuging, platelets are passed through a porous woven or non-woven media filter, which may have a modified surface, to remove white blood cells. However, use of the porous filter introduces its own set of problems. Conventional porous filters may be inefficient because they may permanently remove or trap approximately 5-20% of the platelets. These conventional filters may also reduce “platelet viability” meaning that once passed through a filter a percentage of the platelets cease to function properly and may be partially or fully activated. In addition, porous filters may cause the release of bradykinin, an inflammation mediator and vasodialator, which may lead to hypotensive episodes in a patient. Porous filters are also expensive and often require additional time-consuming manual labor to perform a filtration process. Although porous filters are effective in removing a substantial number of white blood cells, activated platelets may clog the filter. Therefore, the use of at least some porous filters is not feasible in on-line processes.  
      Another separation process is one known as centrifugal elutriation. This process separates cells suspended in a liquid medium without the use of a membrane filter. In one common form of elutriation, a cell batch is introduced into a flow of liquid elutriation buffer. This liquid, which carries the cell batch in suspension, is then introduced into a funnel-shaped chamber located on a spinning centrifuge. As additional liquid buffer solution of a given density flows through the chamber, the liquid sweeps smaller sized, slower-sedimenting cells toward an elutriation boundary within the chamber, while larger, faster-sedimenting cells migrate to an area of the chamber having the greatest centrifugal force.  
      When the centrifugal force and force generated by the fluid flow are balanced, the fluid flow is increased to force slower-sedimenting cells from an exit port in the chamber, while faster-sedimenting cells are retained in the chamber. If fluid flow through the chamber is increased, progressively larger, faster-sedimenting cells may be removed from the chamber.  
      Thus, centrifugal processing separates particles having different sedimentation velocities. Stoke&#39;s law describes sedimentation velocity (V S ) of a spherical particle as follows: 
 
 V   S =((( D   2   cell *(ρ cell −ρ medium ))/(18*μ medium ))*ω 2   r  
 
 where D is the diameter of the cell or particle, ρ cell  is the density of the particle, ρ medium  is the density of the liquid medium, μ medium  is the viscosity of the medium, and ω is the angular velocity and r is the distance from the center of rotation to the cell or particle. Because the diameter of a particle is raised to the second power in Stoke&#39;s equation and the density of the particle is not, the size of a cell, rather than its density, greatly influences its sedimentation rate. This explains why larger particles generally remain in a chamber during centrifugal processing, while smaller particles are released, if the particles have similar densities. 
 
      As described in U.S. Pat. No. 3,825,175 to Sartory, centrifugal elutriation has a number of limitations. In most of these processes, particles must be introduced within a flow of fluid medium in separate, discontinuous batches to allow for sufficient particle separation. Thus, some elutriation processes only permit separation in particle batches and require an additional fluid medium to transport particles. In addition, flow forces must be precisely balanced against centrifugal force to allow for proper particle segregation.  
      For these and other reasons, there is a need to improve particle separation and/or separation of components of a fluid.  
     SUMMARY OF THE INVENTION  
      The present invention comprises a centrifuge for separating particles suspended in a fluid, particularly blood and blood components, and methods for controlling the centrifuge. The apparatus has a fluid separation chamber mounted on a rotor, the fluid separation chamber having a fluid inlet and a fluid outlet, the fluid inlet being radially outward from the fluid outlet, a first frustro-conical segment adjacent the fluid inlet and radially inward therefrom, a second frustro-conical segment immediately adjacent the first frustro-conical segment and radially inward therefrom, the second frustro conical segment having a taper such that particles within the second frustro-conical segment are subjected to substantially equal and opposite centrifugal and fluid flow forces. The taper of the second frustro-conical segment is selected based on the expected size of particles and expected flow rates, such that at least particles of the average size of expected particles will be subjected to substantially equal and opposite centripetal and fluid forces. The taper may be at least 2.8°, more preferably about 3.0°, such that particles having a size greater than the average size of expected particles will be subjected to such equal and opposite forces. Preferably, the first frustro-conical segment has a greater taper than the second frustro-conical segment.  
      The apparatus may further comprise at least one pump controlling a rate of fluid flow through the fluid separation chamber, a camera configured to observe fluid flow with respect to the fluid separation chamber, and a controller receiving signals from the camera and controlling the motor and the pump. Particles, such as white blood cells, are selectively captured within the second frustro-conical segment in said fluid separation chamber and flushed out of the fluid separation chamber. The quantity of particles captured within said second frustro-conical segment may be determined using data derived from the camera. In addition, a limited quantity of relatively high density particles, such as red blood cells, may be captured within the first frustro-conical segment before capturing relatively low density particles, such as white blood cells, within the second frustro-conical segment.  
      It is to be understood that both the foregoing general description and the following detailed description are exemplary, and are intended to provide further explanation of the invention as claimed. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  is a partial perspective view of a centrifuge apparatus including a fluid chamber in accordance with an embodiment of the invention.  
       FIG. 2  is a partial perspective, schematic view of the centrifuge apparatus and a control camera.  
       FIG. 3  is a perspective view of a blood processing apparatus with control camera and lighting.  
       FIG. 4  is a top plan view of the blood processing apparatus of  FIG. 3 .  
       FIG. 5  is a partial cross-sectional view of blood processing apparatus of  FIG. 4  including the centrifuge and fluid chamber of  FIG. 1 .  
       FIG. 6  is a partial cross-sectional, schematic view of a portion of a separation vessel and the fluid chamber mounted on a centrifuge rotor of  FIG. 1 .  
       FIG. 7  is an exploded plan view of the fluid chamber of  FIG. 1 .  
       FIG. 8  is a cross-sectional view of the fluid chamber of  FIG. 7 .  
       FIG. 9  is a perspective view of a tubing set including the fluid chamber and an alternative embodiment of the separation vessel.  
       FIG. 10  is a flow chart of steps for processing blood in the blood processing apparatus.  
       FIG. 11  is a plan view of a separation chamber of the separation vessel of  FIGS. 6 and 9 .  
       FIG. 12  is a partial perspective, schematic view of an alternative centrifuge apparatus and a two control cameras.  
       FIG. 13  is a cross-sectional plan view of the fluid chamber of  FIG. 1 . 
    
    
     DETAILED DESCRIPTION  
      To describe the present invention, reference will now be made to the accompanying drawings.  
      The present invention preferably comprises a blood processing apparatus having a camera control system, as disclosed in U.S. patent applications Ser. Nos. 10/884,877 and 10/905,353. It may also be practiced with a TRIMA® blood component centrifuge manufactured by Gambro BCT, Inc. of Colorado or, alternatively, with a COBE® SPECTRA™ single-stage blood component centrifuge also manufactured by Gambro BCT, Inc. Both the TRIMA® and the SPECTRA™ centrifuges incorporate a one-omega/two-omega sealless tubing connection as disclosed in U.S. Pat. No. 4,425,112 to Ito, the entire disclosure of which is incorporated herein by reference. The SPECTRA™ centrifuge also uses a single-stage blood component separation channel substantially as disclosed in U.S. Pat. No. 4,094,461 to Kellogg et al. and U.S. Pat. No. 4,647,279 to Mulzet et al., the entire disclosures of which are also incorporated herein by reference. The invention could also be practiced with a TRIMA® or TRIMA ACCEL® centrifugal separation system or other types of centrifugal separator. The method of the invention is described in connection with the aforementioned blood processing apparatus and camera control system for purposes of discussion only, and this is not intended to limit the invention in any sense.  
      As embodied herein and illustrated in  FIG. 1 , a centrifuge apparatus  10  has a centrifuge rotor  12  coupled to a motor  14  so that the centrifuge rotor  12  rotates about its axis of rotation A-A. The rotor  12  has a retainer  16  including a passageway or annular groove  18  having an open upper surface adapted to receive a separation vessel  28 , shown in  FIG. 9 . The groove  18  completely surrounds the rotor&#39;s axis of rotation A-A and is bounded by an inner wall  20  and an outer wall  22  spaced apart from one another to define the groove  18  therebetween. Although the groove  18  shown in  FIG. 1  completely surrounds the axis of rotation A-A, the groove could partially surround the axis A-A when the separation vessel is not annular.  
      Preferably, a substantial portion of the groove  18  has a constant radius of curvature about the axis of rotation A-A and is positioned at a maximum possible radial distance on the rotor  12 . This shape ensures that substances separated in the separation vessel  28  undergo relatively constant centrifugal forces as they pass from an inlet portion to an outlet portion of the separation vessel  28 . The motor  14  is coupled to the rotor  12  directly or indirectly through a shaft  24  connected to the rotor  12 . Alternately, the shaft  24  may be coupled to the motor  14  through a gearing transmission (not shown).  
      As shown in  FIG. 1 , a bracket  26  is provided on a top surface of the rotor  12 . The bracket  26  releasably holds a fluid chamber  30  on the rotor  12  so that an outlet  32  of the fluid chamber  30  is positioned closer to the axis of rotation A-A than an inlet  34  of the fluid chamber  30 . The bracket  26  preferably orients the fluid chamber  30  on the rotor  12  with a longitudinal axis of the fluid chamber  30  in a plane transverse to the rotor&#39;s axis of rotation A-A. In addition, the bracket  26  is preferably arranged to hold the fluid chamber  30  on the rotor  12  with the fluid chamber outlet  32  facing the axis of rotation A-A. Although the fluid chamber  30  is shown on a top surface of the rotor  12 , the fluid chamber  30  could also be secured to the rotor  12  at alternate locations, such as beneath the top surface of the rotor  12 .  
       FIG. 2  schematically illustrates an exemplary embodiment of an optical monitoring system  40  capable of measuring a distribution of scattered and/or transmitted light intensities corresponding to patterns of light originating from an observation region on the separation vessel  28 . The monitoring system  40  comprises light source  42 , light collection element  44 , and detector  46 . Light source  42  is in optical communication with the centrifuge apparatus  10  comprising rotor  12 , which rotates about central rotation axis A-A. Rotation about central rotation axis A-A results in separation of a blood sample in the separation vessel  28  into discrete blood components along a plurality of rotating separation axes oriented orthogonal to the central rotation axis A-A.  
      Light source  42  provides incident light beam  54 , which stroboscopically illuminates an observation region  58  when the observation region  58  passes under the light collection element  44 . Light source  42  is capable of generating an incident light beam, a portion of which is transmitted through at least one blood component undergoing separation in separation vessel  28 . At least a portion of scattered and/or transmitted light  56  from the observation region  58  is collected by light collection element  44 . Light collection element  44  is capable of directing at least a portion of the collected light  56  onto detector  46 . The detector  46  detects patterns of scattered and/or transmitted light  56  from the observation region, thereby measuring distributions of scattered and/or transmitted light intensities. Distributions of scattered and/or transmitted light intensities comprise images corresponding to patterns of light originating from the observation region  58 . The images may be monochrome images, which provide a measurement of the brightness of separated blood components along the separation axis. Alternatively, the images may be color images, which provide a measurement of the colors of separated blood components along the separation axis.  
      Observation region  58  is positioned on a portion of the density centrifuge  10 , preferably on the separation vessel  28 . The fluid chamber  30  may also be an observation region, as explained below. In the exemplary embodiment illustrated in  FIG. 6 , separated blood components and phase boundaries between optically differentiable blood components are viewable in observation region  58 . Optionally, the observation region  58  may also be illuminated by an upper light source  62 , which is positioned on the same side of the separation chamber as the light collection element  44  and detector  46 . Upper light source  62  is positioned such that it generates an incident beam  64 , which is scattered by the blood sample and/or centrifuge. A portion of the light from upper light source  62  is collected by light collection element  44  and detected by detector  46 , thereby measuring a distribution of scattered and/or transmitted light intensities.  
      Detector  46  is also capable of generating output signals corresponding to the measured distributions of scattered and/or transmitted light intensities and/or images. The detector  46  is operationally connected to a device controller  60  capable of receiving the output signals. Device controller  60  displays the measured intensity distributions, stores the measured intensity distributions, processes measured intensity distributions in real time, transmits control signals to various optical and mechanical components of the monitoring system and centrifuge or any combination of these. Device controller  60  is operationally connected to centrifuge apparatus  10  and is capable of adjusting selected operating conditions of the centrifuge apparatus, such as the flow rates of cellular and non-cellular components out of the separation vessel  28  or fluid chamber  30 , the position of one or more phase boundaries, rotational velocity of the rotor about central rotation axis A-A, the infusion of anticoagulation agents or other blood processing agents to the blood sample, or any combination of these.  
      Device controller  60  can also be operationally connected to light source  42  and/or upper light source  62 . Device controller  60  and/or detector  46  are capable of generating output signals for controlling illumination conditions. For example, output signals from the detector  46  can be used to control the timing of illumination pulses, illumination intensities, the distribution of illumination wavelengths and/or position of light source  42  and/or upper light source  62 . Device controller  60  and detector  46  are in two-way communication, and the device controller sends control signals to detector  46  to selectively adjust detector exposure time, detector gain and to switch between monochrome and color imaging.  
      Light collection element  44 , detector  46 , or both, can be arranged such that they are moveable, for example moveable along a first detection axis D-D, which is oriented orthogonal to the central rotation axis of the centrifuge. Movement of light collection element  44  in a direction along detection axis D-D adjusts the position of observation region  58  on the density centrifuge. In another embodiment, light collection element  44  is also capable of movement in a direction along a second detection axis (not shown), which is orthogonal to the first detection axis D-D. The present invention also includes an embodiment wherein light source  42 , upper light source  62 , or both, are also capable of movement in a manner to optimize illumination and subsequent detection of transmitted and/or scattered light from the selectively adjustable observation region.  
      Light sources comprise light emitting diode sources capable of generating one or more incident beams for illuminating an observation region on the centrifuge. A plurality of lamps may be positioned to illuminate a single side or multiple sides of the centrifuge apparatus  10 . Light emitting diodes and arrays of light emitting diode light sources are preferred for some applications because they are capable of generating precisely timed illumination pulses. Preferred light sources generate an incident light beam having a substantially uniform intensity, and a selected wavelength range.  
      The optical monitoring system comprises a plurality of light sources, each capable of generating an incident light beam having a different wavelength range, for example, a combination of any of the following: white light source, red light source, green light source, blue light source and infra red light source. Use of a combination of light sources having different wavelength ranges is beneficial for discriminating and characterizing separated blood fractions because absorption constants and scattering coefficients of cellular and non-cellular components of blood vary with wavelength. For example, a component containing red blood cells is easily distinguished from platelet-enriched plasma by illumination with light having wavelengths selected over the range of about 500 nm to about 600 nm, because the red blood cell component absorbs light over this wavelength significantly more strongly that the platelet-enriched plasma component. In addition, use of multiple colored light sources provides a means of characterizing the white blood cell type in an extracted blood component. As different white blood cell types have different absorption and scattering cross sections at different wavelengths, monitoring transmitted and/or scattered light from a white cell-containing blood component provides a means of distinguishing the various white blood cell types in a blood component and quantifying the abundance of each cell-type.  
      The light sources provide a continuous incident light beam or a pulsed incident light beam. Pulsed light sources are switched on and off synchronously with the rotation of the rotor to illuminate an observation region having a substantially fixed position on the rotor. Alternatively, pulsed light sources of the present invention can be configured such that they can be switched on and off in a manner asynchronous with the rotation of the rotor, illuminating different observation regions for each full rotation. This alternative embodiment provides a method of selectively adjusting the location of the observation region and, thereby, probing different regions of the separation chamber or of the fluid chamber  30 . Triggering of illumination pulses may be based on the rotational speed of the centrifuge or on the angular position of the separation chamber or the fluid chamber  30  as detected by optical or electronic methods well known in the art. Triggering may be provided by trigger pulses generated by the device controller  60  and/or detector  46 .  
       FIG. 3  is a perspective side view of the optical monitoring system  40 .  FIG. 4  is a top plan view of the optical monitoring system.  FIG. 5  is a cutaway view corresponding to cutaway line  5 - 5  indicated in  FIG. 4 . The illustrated optical monitoring system  40  comprises CCD camera  72  equipped with a fixed focus lens system (corresponding to the light collection element  44  and detector  46 ), an optical cell  74  (corresponding to the observation region  58 ), an upper LED light source  76  (corresponding to the upper light source  62 ), and a bottom pulsed LED light source  78  (corresponding to the light source  42 ). As illustrated in  FIG. 5 , CCD camera  72  is in optical communication with optical cell  74  and positioned to intersect optical axis  80 . Upper LED light source  76  is in optical communication with optical cell  74  and is positioned such that it is capable of directing a plurality of collimated upper light beams  82 , propagating along propagation axes that intersect optical axis  80 , onto the top side  84  of optical cell  74 . Bottom pulsed LED light source  78  is also in optical communication with optical cell  74  and is positioned such that it is capable of directing a plurality of collimated bottom light beams  86 , propagating along optical axis  80 , onto the bottom side  88  of optical cell  74 .  
      CCD camera  72  may be positioned such that the focal plane of the fixed focus lens system is substantially co-planar with selected optical surfaces of optical cell  74 , such as optical surfaces corresponding to an interface monitoring region, calibration markers, one or more extraction ports and one or more inlets. The CCD camera  72  is separated from the center of the fixed focus lens system by a distance along optical axis  80  such that an image corresponding to selected optical surfaces of optical cell  74  is provided on the sensing surface of the CCD camera. This optical configuration allows distributions of light intensities comprising images of rotating optical cell  74  or of fluid chamber  30  to be measured and analyzed in real time.  
      Mounting assembly  90  holds CCD camera  72  in a fixed position. The mounting assembly  90 , shown in  FIGS. 3 and 4 , comprises a bracket capable of maintaining a fixed position and orientation of CCD camera  72 . Mounting assembly  90  can also comprise a two-axis locking translation stage, optionally with a two-axis tilting mechanism, capable of selectively adjusting the relative orientation and position of the camera with respect to optical cell  74  or fluid chamber  30 . As shown in  FIGS. 3-5 , optical monitoring system  40  is integrated directly into a centrifuge apparatus  10 . To provide good mechanical stability for optical monitoring system  40 , mounting assembly  90  is directly affixed to a frame member (not shown in  FIGS. 3-5 ) supporting housing  92  of centrifuge apparatus  10 . Bottom LED light source  78  is also affixed to a frame member (not shown in  FIGS. 3-5 ) supporting housing  92  of density centrifuge blood processing device  10  by means of an additional mounting assembly  94 . Upper LED light source  76  is secured to CCD camera  72 , as shown in  FIGS. 3-4 . Alternatively, upper LED light source  76  can be directly affixed to a frame member supporting housing  92  of the blood processing device by means of an additional mounting assembly. Mounting assemblies useful in the present invention comprise any fastening means known in the art, such as clamps, brackets, connectors, couplers, additional housing elements and all known equivalents, and can be affixed to frame members supporting housing  92  by any means known in the art including the use of bolts, fasteners, clamps, screws, rivets, seals, joints, couplers or any equivalents of these known in the art.  
      Referring to the cross section shown in  FIG. 5 , first transparent plate  96  is provided between CCD camera  72  and optical cell  74 , and second transparent plate  98  is provided between bottom LED light source  78  and optical cell  74 . First and second transparent plates  96  and  98  physically isolate CCD camera  72 , upper LED light source  76  and bottom LED light source  78  from optical cell  74  so that these components will not contact a sample undergoing processing in the event of sample leakage from the separation chamber. In addition, first and second transparent plates  96  and  98  minimize degradation of CCD camera  72 , upper LED light source  76  and bottom LED light source  78  due to unwanted deposition of dust and other contaminants that can be introduced to the system upon rotation of the separation chamber and filler. Further, first and second transparent plates  96  and  98  also allow a user to optimize the alignment of the camera, upper LED light source and bottom LED light source without exposure to a blood sample in the separation chamber. First and second transparent plates  96  and  98  can comprise any material capable of transmitting at least a portion of upper and bottom illumination light beams  82  and  86 . Exemplary materials for first and second transparent plates  96  and  98  include, but are not limited to, glasses such as optical quality scratch resistant glass, transparent polymeric materials such as transparent plastics, quartz and inorganic salts.  
       FIG. 6  schematically illustrates a portion of the separation vessel  28  and fluid chamber  30  mounted on the rotor  12 . The separation vessel  28  has a generally annular flow path  100  and includes an inlet portion  102  and outlet portion  104 . A wall  106  prevents substances from passing directly between the inlet and outlet portions  102  and  104  without first flowing around the generally annular flow path  100  (e.g., counterclockwise in  FIG. 6 ).  
      A radial outer wall  108  of the separation vessel  28  is positioned closer to the axis of rotation A-A in the inlet portion  102  than in the outlet portion  104 . During separation of blood components, this arrangement causes formation of a very thin and rapidly advancing red blood cell bed in the separation vessel  28  between the inlet portion  102  and outlet portion  104 . The red blood cell bed reduces the amount of blood components required to initiate a separation procedure, and also decreases the number of unnecessary red blood cells in the separation vessel  28 . The red blood cell bed substantially limits or prevents platelets from contacting the radial outer wall  108  of the separation vessel  28 . This is believed to reduce clumping of platelets caused when platelets contact structural components of centrifugal separation devices.  
      The inlet portion  102  includes an inflow tube  110  for conveying a fluid to be separated, such as whole blood, into the separation vessel  28 . During a separation procedure, substances entering the inlet portion  102  follow the flow path  100  and stratify according to differences in density in response to rotation of the rotor  12 . The outlet portion  104  includes first, second, and third outlet lines  112 ,  114 ,  116  for removing separated substances from the separation vessel  28 . Preferably, each of the components separated in the vessel  28  is collected and removed in only one area of the vessel  28 , namely the outlet portion  104 . In addition, the separation vessel  28  preferably includes a substantially constant radius except in the region of the outlet portion  104  where the outer wall of the outlet portion  104  is preferably positioned farther away from the axis of rotation A-A to allow for outlet ports of the lines  112 ,  114 , and  116  to be positioned at different radial distances and to create a collection pool with greater depth for the high density red blood cells. The outlet port of line  114  is farther from the axis of rotation A-A than the other ports to remove higher density components, such as red blood cells. The port of line  116  is located closer to the axis of rotation A-A than the other ports to remove the least dense components separated in the separation vessel  28 , such as plasma. The first line  112  collects intermediate density components and, optionally, some of the lower density components. The second and third lines  114  and  116  are positioned downstream from first line  112  to collect the high and low density components.  
      The positions of the interfaces are controlled by the CCD camera  72  monitoring the position of the interface and controlling flow of liquid and/or particles in response to the monitored position. Further details concerning the structure and operation of the separation vessel  28  are described in U.S. patent application Ser. No. 10/884,877 and also in U.S. Pat. No. 4,094,461 to Kellogg et al. and U.S. Pat. No. 4,647,279 to Mulzet et al., which have been incorporated herein by reference.  
      A ridge  144  extends from the inner wall  20  of the groove  18  toward the outer wall  22  of the groove  18 . When the separation vessel  28  is loaded in the groove  18 , the ridge  144  deforms semi-rigid or flexible material in the outlet portion  104  of the separation vessel  28  to form a trap dam  146  in the separation vessel  28 , upstream from the first line  112 . The trap dam  146  extends away from the axis of rotation A-A to trap a portion of lower density substances, such as priming fluid and/or plasma, along an inner portion of the separation vessel  28  located upstream of the trap dam  146 . These trapped substances help convey platelets to the outlet portion  104  and first line  112  by increasing plasma flow velocities next to the layer of red blood cells in the separation vessel  28  to scrub platelets toward the outlet portion  104 . A downstream portion  148  of the trap dam  146  has a relatively gradual slope extending in the downstream direction toward the axis of rotation A-A, which limits the number of platelets (intermediate density components) that become re-entrained (mixed) with plasma (lower density components) as plasma flows along the trap dam  146 . In addition, the gradual slope of the downstream portion  148  reduces the number of platelets that accumulate in the separation vessel  28  before reaching the first collection port of first line  120 .  
      The camera  44  is generally focused on the separation vessel and stroboscopic illumination allows an observation region  58  around the first, second, and third lines  112 ,  114 , and  116  to be observed. Using information gathered through the camera, the controller  60  regulates the position of interfaces between various blood components, such as plasma, buffy coat (containing monocytes and/or white blood cells and platelets) and red blood cells by controlling the pumps  158 ,  160 , and  162 .  FIG. 11  shows an image of the observation region  58  generated by the methods of U.S. patent application Ser. No. 10/884,877 (incorporated herein by reference) corresponding to the separation of a human blood sample and extraction of a separated white blood cell-containing blood component. The observation region  58  shown in  FIG. 11  includes a phase boundary monitoring region  202  and a white blood cell extraction port monitoring region  204 . Visible in phase boundary monitoring region  202  are a red blood cell component  206 , a plasma component  208  and a mixed-phase buffy coat layer  210 , which has both white blood cells and platelets. Several calibration markers are also apparent in the image in  FIG. 11 . The edge  212  of the optical cell comprises a first calibration marker for determining the absolute position of phase boundaries between optically differentiable blood components. A series of bars  214  having a thickness of 1 mm and known scattering and absorption characteristics comprises a second calibration marker useful for optimizing the focusing of the light collection element and indicating the positions and physical dimensions of the phase boundary monitoring region  202  and the white blood cell extraction port monitoring region  204 . Light intensities transmitted through the phase boundary monitoring region  202  are acquired as a function of time and analyzed in real time to provide measurements of the position of the phase boundary  216  between red blood cell component  206  and buffy coat layer  210  and the phase boundary  218  between the buffy coat layer  210  and plasma component  208 . All boundary layer positions are measured relative to the edge of the optical cell  212 .  
      White blood cell extraction port monitoring region  204  includes a first flux monitoring region  220  and a second flux monitoring region  222  positioned on first line  112  of the optical cell for extracting white blood cells. In this example, first line  112  having orifice  224  is configured to collect white blood cells in the human blood sample and extends a distance along the separation axis of such that it terminates proximate to the buffy coat layer in the rotating separation chamber. The two-dimensional distribution of light intensities of light transmitted through the first and second flux monitoring regions  220  and  222  depends on the concentration, and spatial distribution and cell-type of cellular material exiting the separation chamber. Light intensities transmitted through and reflected from first and second flux monitoring regions  220  and  222  were acquired as a function of time and analyzed to characterize the composition and flux of cellular material out of the separation chamber. As cellular materials, such as white blood cells and red blood cells, absorb and scatter light from the light sources, passage of cellular material through the extraction port decreases the observed light intensities.  
      Referring again to  FIG. 6 , the outer wall  22  of the groove  18  preferably includes a gradual sloped portion  152  facing the ridge  144  in the inner wall  20 . When the separation vessel  28  shown in  FIG. 9  is loaded in the groove  18 , the gradual sloped portion  152  deforms semi-rigid or flexible material in the outlet portion  104  of the separation vessel  28  to form a relatively smooth and gradual sloped segment in a region of the vessel  28  across from the trap dam  146 , which slopes gradually away from the axis of rotation A-A to increase the thickness of a layer of high-density fluid components, such as red blood cells, formed across from the trap dam  146 .  
      The first collection line  112  is connected to the fluid chamber inlet  34  to pass the intermediate density components into the fluid chamber  30 . Components initially separated in the separation vessel  28  are further separated in the fluid chamber  30 . For example, white blood cells could be separated from plasma and platelets in the fluid chamber  30 . This further separation preferably takes place by forming a saturated fluidized bed of particles, such as white blood cells, in the fluid chamber  30 . The fluid chamber  30  may be formed of a transparent or translucent co-polyester plastic, such as PETG, to allow viewing of the contents within the chamber interior with the aid of the camera during a separation procedure.  
      As schematically shown in  FIG. 6 , a plurality of pumps  158 ,  160 , and  162  are provided for adding and removing substances to and from the separation vessel  28  and fluid chamber  30 . An inflow pump  158  is coupled to the inflow line  110  to supply the substance to be separated, such as whole blood, to the inlet portion  102 . In addition, a first collection pump  160  is flow coupled to the outflow tubing  130  connected to the fluid chamber outlet  32 , and a second collection pump  162  is flow coupled to the third collection line  116 . The first collection pump  160  draws liquid and particles from the fluid chamber outlet  32  and causes liquid and particles to enter the fluid chamber  30  via the fluid chamber inlet  34 . The second collection pump  162 , on the other hand, removes primarily low-density substances from the separation vessel  28  via the third line  116 .  
      The pumps  158 ,  160 , and  162  are peristaltic pumps or impeller pumps configured to prevent significant damage to blood components. However, any fluid pumping or drawing device may be provided. In an alternative embodiment (not shown), the first collection pump  160  may be fluidly connected to the fluid chamber inlet  34  to directly move substances into and through the fluid chamber  30 . In addition, the pumps  158 ,  160 , and  162  may be mounted at any convenient location. The inflow pump  158  and the first collection pump  160  may be configured so that substances do not bypass these pumps when they are paused. For example, when the first collection pump  160  is temporarily paused, substances pumped by the second collection pump  162  flow into the fluid chamber outlet  32  rather than bypassing the pump  160  and flowing in the opposite direction.  
      The apparatus  10  further includes a controller  164  ( FIG. 1 ) connected to the motor  14  to control rotational speed of the rotor  12 . The controller  164  is connected to the pumps  158 , 160 , and  162  to control the flow rate of substances flowing to and from the separation vessel  28  and the fluid chamber  30 . The controller  164  maintains a saturated fluidized bed of first particles within the fluid chamber  30  to aid in second particles being retained in the fluid chamber  30 . The controller  164  also preferably controls the operation and flow rate of the pumps  158 ,  160 ,  162  to permit the temporary purging of the fluid chamber  30 . The controller  164  may include a computer having programmed instructions provided by a ROM or RAM as is commonly known in the art. The controller  164  may vary the rotational speed of the centrifuge rotor  12  by regulating frequency, current, or voltage of the electricity applied to the motor  14 . Alternatively, the rotational speed can be varied by shifting the arrangement of a transmission (not shown), such as by changing gearing to alter a rotational coupling between the motor  14  and rotor  12 . The controller  164  may receive input from a rotational speed detector (not shown) to constantly monitor the rotation speed of the rotor.  
      After loading the separation vessel  28  and fluid chamber  30  on the rotor  12 , the separation vessel  28  and chamber  30  are initially primed with a low density fluid medium, such as air, saline solution, plasma, or another fluid substance having a density less than or equal to the density of liquid plasma. Alternatively, the priming fluid is whole blood itself. This priming fluid allows for efficient establishment of a saturated fluidized bed of red blood cells within the fluid chamber  30 . When saline solution is used, the pump  158  pumps this priming fluid through the inflow line  110  and into the separation vessel  28  via the inlet line  110 . The saline solution flows from the inlet portion  102  to the outlet portion  104  (counterclockwise in  FIG. 6 ) and through the fluid chamber  30  when the controller  164  activates the pump  160 . Controller  164  also initiates operation of the motor  14  to rotate the centrifuge rotor  12 , separation vessel  28 , and fluid chamber  30  about the axis of rotation A-A. During rotation, twisting of lines  110 ,  112 ,  114 ,  116 , and  130  is prevented by a sealless one-omega/two-omega tubing connection as is known in the art and described in above-mentioned U.S. Pat. No. 4,425,112.  
      As the separation vessel  28  rotates, a portion of the priming fluid (blood or saline solution) becomes trapped upstream from the trap dam  146  and forms a dome of priming fluid (plasma or saline solution) along an inner wall of the separation vessel  28  upstream from the trap dam  146 . After the apparatus  10  is primed, and as the rotor  12  rotates, whole blood or blood components are introduced into the separation vessel  28 . When whole blood is used, the whole blood can be added to the separation vessel  28  by transferring the blood directly from a donor or patient through inflow line  110 . In the alternative, the blood may be transferred from a container, such as a blood bag, to inflow line  110 .  
      The blood within the separation vessel  28  is subjected to centrifugal force causing components of the blood components to separate. The components of whole blood stratify in order of decreasing density as follows: (1) red blood cells, (2) white blood cells, (3) platelets, and (4) plasma. The controller  164  regulates the rotational speed of the centrifuge rotor  12  to ensure that this particle stratification takes place. A layer of red blood cells (high density component(s)) forms along the outer wall of the separation vessel  28  and a layer of plasma (lower density component(s)) forms along the inner wall of the separation vessel  28 . Between these two layers, the intermediate density platelets and white blood cells (intermediate density components) form a buffy coat layer. This separation takes place while the components flow from the inlet portion  102  to the outlet portion  104 . Preferably, the radius of the flow path  100  between the inlet and outlet portions  102  and  104  is substantially constant to maintain a steady red blood cell bed in the outlet portion  104  even if flow changes occur.  
      In the outlet portion  104 , platelet poor plasma flows through the third line  116 . These relatively low-density substances are pumped by the second collection pump  162  through the third collection line  116 . Red blood cells are removed via the second line  114 . The red blood cells flow through the second collection line  114  and can then be collected and optionally recombined with other blood components or further separated. Alternately, these removed blood components may be re-infused into a donor or patient.  
      Accumulated platelets are removed via the first collection line  112  along with some of the white blood cells and plasma. As the platelets, plasma, white blood cells, and possibly a small number or red blood cells pass through the first collection line  112 , these components flow into the fluid chamber  30 , filled with the priming fluid, so that a saturated fluidized particle bed may be formed. The portion or dome of priming fluid (i.e. saline) trapped along the inner wall of the separation vessel  28  upstream from the trap dam  146  guides platelets so that they flow toward the first collection line  112 . The trapped fluid reduces the effective passageway volume and area in the separation vessel  28  and thereby decreases the amount of blood initially required to prime the system in a separation process. The reduced volume and area also induces higher plasma and platelet velocities next to the stratified layer of red blood cells, in particular, to “scrub” platelets toward the first collection line  112 . The rapid conveyance of platelets increases the efficiency of collection.  
      The controller  164  maintains the rotation speed of the rotor  12  within a predetermined rotational speed range to facilitate formation of this saturated fluidized bed. In addition, the controller  164  regulates the pump  160  to convey at least the plasma, platelets, and white blood cells at a predetermined flow rate through the first collection line  112  and into the inlet  34  of the fluid chamber  30 . These flowing blood components displace the priming fluid from the fluid chamber  30 . When the platelet and white blood cell particles enter the fluid chamber  30 , they are subjected to two opposing forces. Plasma flowing through the fluid chamber  30  with the aid of pump  160  establishes a first viscous drag force when plasma flowing through the fluid chamber  30  urges the particles toward the outlet  32 . A second centrifugal force created by rotation of the rotor  12  and fluid chamber  30  acts to urge the particles toward the inlet  34 .  
      The controller  164  regulates the rotational speed of the rotor  12  and the flow rate of the pump  160  to collect platelets and white blood cells in the fluid chamber  30 . As plasma flows through the fluid chamber  30 , the flow velocity of the plasma decreases and reaches a minimum as the plasma flow approaches the maximum cross-sectional area of the fluid chamber  30 . Because the rotating centrifuge rotor  12  creates a sufficient gravitational field in the fluid chamber  30 , the platelets accumulate near the maximum cross-sectional area of the chamber  30 , rather than flowing from the chamber  30  with the plasma. The white blood cells accumulate somewhat radially outward from the maximum cross-sectional area of the chamber  30 . However, density inversion tends to mix these particles slightly during this initial establishment of the saturated fluidized particle bed.  
      The fluid chamber  30  is configured to allow cyclic collection of selected particles, such as white blood cells, followed by efficient evacuation of the cells into a collection bag. In contrast to other chamber designs for forming saturated fluidized beds, the fluid chamber described herein has particular application for the automated collection of blood components in that a bolus of cells having selected characteristics can be collected in the fluid chamber  30  and then flushed with low density fluid into a collection bag and these steps can be repeated multiple times, allowing a larger quantity of the selected cells to be collected from the donor or patient while reducing the amount of time necessary for the donation process. Collection of cells in the fluid chamber can be monitored by the camera  72  and the device controller  60 . When a selected quantity of cells have been collected in the fluid chamber  30 , the flow of plasma through the chamber can be increased and gravity force reduced and the collected cells can be washed out of the chamber and directed into a collection bag.  
      The fluid chamber  30  may be constructed in two pieces, a main body  166  and a cap  168 , both being symmetrical around an axis  170 . The main body  166  has an inlet  34  comprising a through bore  172  and a concentric stopped bore  174 . The diameter of the through bore  172  corresponds to the inside diameter of the first outlet line  112 , while the diameter of the stopped bore  174  corresponds to the outside diameter of the first outlet line  112 , so that the outlet line  112  can be seated in the stopped bore  174  and a fluid passageway of constant diameter can be formed between the outlet line  112  and the through bore  172 . The through bore  172  opens into a first frustro-conical segment  176 . A wall  178  of the first frustro-conical segment  176  tapers away from the axis  170  at an angle of about 16°. Immediately adjacent to and down stream from the first frustro-conical segment  176 , a second frustro-conical segment  180  extends from the first frustro-conical segment  176  to a distal end  182  of the main body  166 . A wall  184  of the second frustro-conical segment  180  tapers away from the axis  170  at an angle of about 3°. As blood components such as plasma, platelets and white blood cells flow into the fluid chamber  30 , they are affected by rotational speed, fluid flow rate, and the configuration of the fluid chamber. For example, in a frustro-conical segment, fluid flow rate will decrease as the cross sectional area of the segment increases. At the same time, the blood components may be subject to a centripetal force resulting from the rotation of the apparatus. The centripetal force experienced by a particle in the segment will decrease as the particle moves radially inward toward the axis of rotation. With the proper configuration, a balance of change in forces can be attained such that decreased centripetal force as a particle moves inward is balanced by a corresponding decrease in force of fluid flow. The sizes of white blood cells are distributed about an average size. It has been determined that, for the average size of white blood cells, a increase in cross sectional area represented by a 2.8° taper in the second frustro-conical segment  180  balances the mentioned forces and creates a relatively large area within the fluid chamber  30  where the forces acting on a particle are relatively constant. A slightly larger taper, for example 3° taper, captures slightly larger cells as well, and should be used for that reason. In contrast to the second segment  180 , the first segment  176  has a steeper angle and particles in this region are more affected by the change in cross-sectional area than by the change in centripetal force. Particles are pushed through the first segment by fluid flow, gradually slowing as the flow rate diminishes. In the second segment  180 , the particles experience substantially constant forces. By altering either the rate of rotation or the fluid flow rate or both the countervailing forces of fluid pushing in and centripetal force pushing out can be balanced for the particular particle of interest. The selected particles begin to enter the fluid chamber  30 . Using the camera and techniques explained in U.S. patent application Ser. No. 10/884,877, the flux of cells passing into the fluid chamber  30  can be measured and the controller  60  can calculate the number of blood cells captured in the fluid chamber. Initially, the boundary  216  between red blood cells and the buffy coat can be raised and a few red blood cells can be drawn into the fluid chamber  30 . Because of their weight, the red cells collect in the first segment  176 , where they form a fluidized bed  226 , as shown in  FIG. 13 . The boundary  216  is then lowered and white cells and plasma are drawn into the fluid chamber  30 . As these cells (white blood cells or monocytes) pass through the bed  226  of red cells, the flow velocity across the second segment becomes more uniform across the entire cross-section of the chamber  30 . A relatively flat velocity distribution makes it more likely that the desired cells will be captured in the second segment  180 . Captured white blood cells begin to form a bolus  228 . When the second segment is sufficiently filled with the desired particle, such as white blood cells, the rate of plasma extraction through line  116  can be reduced, for example, from 40 mL/min to 38 mL/min, to lower the interface  218  between plasma and the buffy coat, that is, to move the interface radially outward so that the first outlet line  112  extracts plasma rather than buffy coat. Plasma flowing through the fluid chamber  30  purges the chamber, leaving a concentrated bolus of white blood cells, washed with the donor&#39;s plasma. After purging, the flow rate through the chamber  30  can be increased to flush or evacuate the accumulated particles into collection bag by manipulating valves to temporarily direct the fluids leaving the fluid chamber into the collection bag. The angular velocity of the rotor  12  is reduced to decrease the centripetal or gravitational force acting on the fluid and particles. At the same time, the speed of second pump  162  is further decreased, for example, to 33 mL/min, and the speed of first pump  160  is increased to flush the collected white blood cells into the collection bag. Because a cycle of collecting cells in the fluid chamber and evacuating the collected cells to the collection bag can be performed multiple times, a relatively large amount of a rarer blood component, such as white blood cells, can be collected from a single donor or patient.  
      The controller  60  implements a procedure  230  shown in  FIG. 10 . As explained above, the procedure  230  begins processing  232  by establishing a fluidized bed of a limited quantity of red blood cells in the first segment  176 . With the fluidized bed established, the level of the interface  216  is reduced and white cells begin to flow into the fluid chamber  30 , and are detected  234  such that the controller  60  can calculate the quantity or number of cells in the fluid chamber. Although the camera views the flow only intermittently because of the stroboscopic lighting, the flow rate through the line  112  is slow (5 mL/min) compared to the rotational speed of the centrifuge (3000 rpm) and the strobe rate of the lighting, so an accurate count of the particles or cells passing through the line  112  can be obtained. The controller  60  determines  236  when a sufficient amount of cells has been collected in the fluid chamber  30 , and then purges  238  the cells by lowering the plasma interface  218  and causing plasma to flow through the collected white cells. After this washing, the cells are flushed  240  from the chamber into a collection bag. When the chamber is empty  242 , the controller determines  244  if a predetermined quantity of cells has been collected and either ends  248  the procedure, or begins  232  to collect another quantity of cells.  
      In the illustrated embodiment, the main body  166  of the fluid chamber  30  further comprises a circumferential flange  186 , which is supported in the holder  26 . The size of the flange may be varied so that different types of fluid chambers can be used in a single centrifuge apparatus. Since certain chambers available from Gambro BCT, Inc. are relatively larger in diameter than the fluid chamber described herein, the flange may be designed to compensate for these differences. A plurality of radial fins  188  is formed proximally from the flange  186 . In this embodiment, the fins serve primarily for stability when the fluid chamber  30  is mounted in an existing holder and also as conduits for plastic material during injection molding of the main body  166 . At the distal end  182  of the main body  166 , a groove  190  secures the cap  168  to the distal end. The cap comprises a rim  191  that fits into the groove  190  and a flange  192  which fits against the distal end of the main body. The cap and main body may be joined by ultrasonic welding, or other suitable technique as known in the art. The cap opens into an abrupt frustro-conical segment  194 . The abrupt segment  194  tapers towards the axis  170 , the inner wall  196  of the abrupt segment  194  forming a 36° angle with the axis  170 . The abrupt segment  194  funnels collected blood components flushed from the second segment  180  into the outlet  32  without excessive turbulence or damage to the blood components. The outlet  34  comprises a through bore  198  and a concentric stopped bore  200 . The diameter of the through bore  198  corresponds to the inside diameter of the outflow tubing  130 , while the diameter of the stopped bore  200  corresponds to the outside diameter of the outflow tubing  130 , so that the outflow tubing  130  can be seated in the stopped bore  200  and a fluid passageway of constant diameter can be formed between the outflow tubing  130  and the through bore  198 . The through bore  198  opens into the frustro-conical segment  194 .  
      The state of the fluids in the fluid chamber  30  can also be monitored by direct observation. An optical window may be provided in the fluid chamber  30 , and the fluid may be monitored by a camera system as described above. A single camera may be automatically re-positioned and re-focused to the desired area of the fluid chamber  30 , and the stroboscopic lights synchronized to the radial position of the fluid chamber  30  rather than to the observation region  58 . Preferably, however, two camera systems might be used, as illustrated in  FIG. 12 . A second light collection element or camera  44 ′ is spaced away from the first camera or light collection element  44  and is focused on the fluid chamber  30 , while the first camera  44  is focused on the observation region  58 . Lights  42  and  62  illuminate the observation region  58  when it passes under the first camera  44 . Lights  42 ′ and  62 ′ illuminate the fluid chamber  30  when it passes under the second camera  44 ′.  
      As with the system described heretofore, the system for observing the fluid chamber  30  comprises a light source  42 ′, light collection element  44 ′, and detector  46 ′. Light source  42 ′ is in optical communication with the centrifuge apparatus  10 . Light source  42 ′ provides incident light beam  54 ′, which illuminates the fluid chamber  30 , preferably in a manner generating scattered and/or transmitted light from the fluid in the chamber. In one embodiment, light source  42 ′ is capable of generating an incident light beam, a portion of which is transmitted through at least one blood component in the fluid chamber  30 . At least a portion of scattered and/or transmitted light  56 ′ from the fluid chamber  30  is collected by light collection element  44 ′. Light collection element  44 ′ is capable of directing at least a portion of the collected light  56 ′ onto detector  46 ′. The detector  46 ′ detects patterns of scattered and/or transmitted light  56 ′ from the fluid chamber  30 , thereby measuring distributions of scattered and/or transmitted light intensities. Distributions of scattered and/or transmitted light intensities comprise images corresponding to patterns of light originating from the fluid chamber  30 . The images may be monochrome images, which provide a measurement of the brightness of separated blood components along the separation axis. Alternatively, the images may be color images, which provide a measurement of the colors of separated blood components along the separation axis.  
      Optionally, the fluid chamber  30  can also be illuminated by an upper light source  62 ′, which is positioned on the same side of the separation chamber as the light collection element  44 ′ and detector  46 ′. Upper light source  62 ′ is positioned such that it generates an incident beam  64 ′, which is scattered by the blood sample and/or centrifuge. A portion of the light from upper light source  62 ′ is collected by light collection element  44 ′ and detected by detector  46 ′, thereby measuring a distribution of scattered and/or transmitted light intensities. Detector  46 ′ is also capable of generating output signals corresponding to the measured distributions of scattered and/or transmitted light intensities and/or images. The detector  46 ′ is operationally connected to the device controller  60 , which operates as explained above.  
      Although the inventive device and method have been described in terms of removing white blood cells and collecting platelets, this description is not to be construed as a limitation on the scope of the invention. The invention may be used to separate any of the particle components of blood from one another or the invention could be used in fields other than blood separation. For example, the saturated fluidized bed may be formed from red blood cells to prevent flow of white blood cells through the fluid chamber  22 , so long as the red blood cells do not clump excessively. Alternatively, the liquid for carrying the particles may be saline or another substitute for plasma. In addition, the invention may be practiced to remove white blood cells or other components from a bone marrow harvest collection or an umbilical cord cell collection harvested following birth. In another aspect, the invention can be practiced to collect T cells, stem cells, or tumor cells. Further, one could practice the invention by filtering or separating particles from fluids unrelated to either blood or biologically related substances.  
      It will be apparent to those skilled in the art that various modifications and variations can be made to the structure and methodology of the present invention without departing from the scope or spirit of the invention. Rather, the invention is intended to cover modifications and variations provided they come within the scope of the following claims and their equivalents.