Patent Abstract:
Disclosed is a separator-concentrator, such as for separating and concentrating platelet-rich-plasma (PRP) from whole blood that is suitable for office use or emergency use for trauma victims. The PRP separator comprises a motorized centrifugal separation assembly and a concentrator assembly. The centrifugal separation assembly comprises a centrifugal drum separator and a motor having a drive axis connected to the centrifugal drum separator. The concentrator assembly comprises a water-removal module for preparing PRP concentrate.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 11/342,749 filed on Jan. 30, 2006, now U.S. Pat. No. 7,824,559; which application claims the benefit under 35 USC 120 of the filing dates of U.S. Provisional Application No. 60/651,050, filed on Feb. 7, 2005, U.S. Provisional Application No. 60/654,718, filed on Feb. 17, 2005, and U.S. Provisional Application No. 60/723,312, filed on Oct. 4, 2005. The entire disclosures of the above applications are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to a device and method for preparing platelet-plasma concentrates with improved wound healing properties for use as a tissue sealant and adhesive. The product has a fully active (un-denatured) fibrinogen concentration that is several times greater than the concentration of fibrinogen in blood and a platelet concentration that is greater than the concentration of platelets in blood. 
     BACKGROUND OF THE INVENTION 
     Blood can be fractionated, and the different fractions of the blood are useful for different medical needs. Under the influence of gravity or centrifugal force, blood spontaneously separates into three layers. At equilibrium, the top low-density layer is a straw-colored clear fluid called plasma. Plasma is a water solution of salts, metabolites, peptides, and many proteins ranging from small (insulin) to very large molecules (complement components). 
     The bottom, high-density layer is a deep red viscous fluid comprising anuclear red blood cells (erythrocytes) specialized for oxygen transport. The red color is imparted by a high concentration of chelated iron or heme that is responsible for the erythrocytes&#39; high specific gravity. The relative volume of whole blood that consists of erythrocytes is called the hematocrit, and in normal human beings this can range from about 37% to about 52% of whole blood. 
     The intermediate layer is the smallest, appearing as a thin white band above the erythrocyte layer and below the plasma layer; this is called the buffy coat. The buffy coat itself has two major components, nucleated leukocytes (white blood cells) and anuclear smaller bodies called platelets (or thrombocytes). Leukocytes confer immunity and contribute to debris scavenging. Platelets seal ruptures in blood vessels to stop bleeding, and deliver growth and wound healing factors to a wound site. Slower speed centrifugation or shorter duration centrifugation permits separation of erythrocytes and leukocytes from plasma, while the smaller platelets remain suspended in the plasma, resulting in platelet rich plasma (PRP). 
     U.S. Pat. No. 5,585,007 identifies methods for making plasma concentrates from whole blood for use in wound healing and as a tissue sealant. This patent is hereby incorporated by reference in its entirety. This device, designed for placement in a medical laboratory or surgical amphitheatre, uses a disposable cartridge for preparing tissue sealant. The device was particularly applicable for stat preparations of autologous tissue sealants. Preparation in the operating room of 5 ml of sealant from 50 ml of patient blood required less than 15 minutes and only one simple operator step. There was no risk of tracking error because preparation could take place in the operating room during the surgical procedure. Chemicals added could be limited to anticoagulant (e.g., citrate) and calcium chloride. The disposable cartridge could fit in the palm of the hand and was hermetically sealed to eliminate possible exposure to patient blood and to ensure sterility. Adhesive and tensile strengths of the product were comparable or superior to pooled blood fibrin sealants made by precipitation methods. Use of antifibrinolytic agents (such as aprotinin) was not necessary because the tissue sealant contained high concentrations of natural inhibitors of fibrinolysis from the patient&#39;s blood. 
     This device used a new sterile disposable cartridge with the separation chambers for each run. Since the device was designed to be used in a normal medical setting with ample power, the permanent components were designed for long-term durability, safety and reliability, and were relatively heavy, using conventional centrifuge motors and accessories. 
     Small, self-contained centrifugal devices for obtaining platelet concentrates from blood are described in commonly assigned, copending application Ser. No. 10/394,828 filed Mar. 21, 2003, the entire contents of which are hereby incorporated by reference. This device separates blood into erythrocyte, plasma and platelet layers and selectively removes the platelet layer as a platelet concentrate, that is, platelets suspended in a minimal amount of plasma. The plasma fraction, being in an unconcentrated form, is not effective as a hemostat or tissue adhesive. 
     Platelet rich plasma is a concentrated platelet product that can be produced from whole blood through commercially available systems, resulting in varying levels of platelet concentration. Platelets play a crucial role in the signaling cascade of normal wound healing. Activated platelets release the contents of their α-granules resulting in a deposition of powerful growth factors such as platelet derived growth factor (PDGF), transforming growth factor β-(TGF-β), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). PRP has been used in many different clinical applications, demonstrating the effectiveness and importance of the product for a variety of medical procedures. For example, percutaneous application of PRP to patients with severe lateral epicondylitis, or tennis elbow, resulted in improved elbow function and reduced pain. Early maturation of bony fusion was observed when platelet concentrate was used during lumbar spinal fusions. Chronic diabetic foot ulcers treated with PRP achieved increased healing rates compared to the control group receiving standard care. Studies by Bhanot el at show decreased formation of hematoma and seroma, decreased postoperative swelling, and improved healing time for plastic surgeries that included PRP in the treatment. Further, during dental surgeries, the use of PRP has improved bone regeneration around implants. 
     PRPs have demonstrated numerous clinical benefits to patients. There are many devices on the market that concentrate platelets to differing levels. At this time, it is unclear the amount of platelets that is most efficient for each surgical application. Concentrations of at least 1,000×10 3  platelets/μL are recommended. The system described in copending application Ser. No. 10/394,828 can provide platelets up to 8 time baseline concentration, and the normal human platelet range is 200×10 3  platelets/μL to 400×10 3  platelets/μL. This means a highly effective concentrate in a range of 1,600×10 3  platelets/μL to 3,200×10 3  platelets/μL. 
     However, the PRP products of the prior invention, while achieving greatly increased platelet concentrations, did not have tissue sealant and hemostatic properties needed for many surgeries. The platelet-free plasma concentrates, while they were excellent sealants and hemostats, did not provide the healing properties of platelets. 
     SUMMARY OF THE INVENTION 
     It is therefore an objective of the present invention to provide an apparatus and method for preparing a novel PRP concentrate that combines enhanced platelet levels in concentrated plasma, in which the fibrinogen levels have not been significantly denatured. 
     The device of this invention is a PRP separator-concentrator comprising a housing, a PRP separation assembly, and a PRP concentration assembly. The concentration assembly has a PRP concentration sump. An axially concentric rigid stationary outlet tube is secured to the housing and extends through the PRP separation assembly to the PRP concentrate sump. The PRP separation assembly is attached to and positioned above the PRP concentration assembly to form a combined separator-concentrator assemblage that is rotatable about the outlet tube. 
     The PRP separation assembly can comprise a separation chamber having an outer wall with an inner wall surface and a sloped floor secured to the outer wall, the inner wall surface being lined with a depth filter having pores and passageways that are sized to receive and entrap erythrocytes during centrifuging. The PRP separation assembly includes a blood inlet. 
     The separation chamber can include a top plate and a balanced distribution of separator plates attached to the outer wall and floor of the separation chamber, the separator plates lying in a plane that is parallel to the central axis. The separator plates can extend from the outer wall radially inward to a distance beyond the surface of the depth filter and from the floor to a position spaced from the top plate. The separation chamber is balanced for substantially vibration-free rotation about the central axis. 
     The PRP concentrator can comprise a concentration chamber having a floor for supporting desiccated beads and a wall with at least one opening closed with a screen. The screen has openings that are sized to retain the desiccated beads in the concentration chamber. The concentration chamber can be surrounded by an outer wall with a sloped floor secured thereto, the sloped floor including at its center, a PRP concentrate sump. The concentrator can have a distribution of upright screen supports, the upright screen supports having an inner surface and an outer surface, the cylindrical screen being supported on the outer surface of the upright screen supports. 
     A stationary bead rake can be secured to the stationary tube and extend outward therefrom, the rake having distal ends that are spaced at a distance from the upright screen supports. The rake can comprise a longitudinal body, the center of which is secured to the rigid outlet tube. The longitudinal body can optionally have weakened fracture points adjacent to the rigid tube, whereby the longitudinal body fractures when it is exposed to excessive strain from swelled bead contact during high speed centrifugation. 
     The concentration assembly can have secured to its bottom, an axially concentric concentrator drive coupling, the PRP separator-concentrator including a motor assembly with a motor coupling that engages the concentrator drive coupling. The motor assembly can comprise a motor control system for timed rotations of the drive coupling during an acceleration phase, a rapid centrifugal erythrocyte separation phase, a deceleration phase, a slow stir concentrating phase, an acceleration phase, and a rapid centrifugal PRP concentrate separation phase. 
     The PRP separator-concentrator of this invention can include a valve assembly and a central passageway connecting the separation chamber and the concentration chamber, the upper surface of the central passageway including a valve seat. The valve seat includes a valve face that forms a seal with the valve seat in the close position and separates to disengage the seal in the open position. The valve assembly can include a pair of opposed normally upright valve operator arms, each operator arm having an inflexible body with a weighted distal end and a flexible proximal end. Each flexible proximal end can be secured to the valve face at a level that elevates the valve face in an axial direction to move the valve face to the open position when the operator arms pivot outward under centrifugal force during fast rotation of the separator-concentrator about its central axis. The flexible proximal ends can be positioned between opposed plates extending upward from the floor of the separation assembly, each plate having plate side edges, the plate side edges being positioned to contact the operator arms and thereby restrain the proximal ends against rotation around the central axis when the arms are in the upright position and to free the operator arms from rotation when the flexible proximal ends are raised above the plate side edges when the valve is opened. The plates can have a top edge that is positioned to support the operator arms after their axial rotation, thereby preventing their return to the upright position when centrifugal rotation is ended, thereby preventing closure of the valve assembly. 
     The method of this invention for preparing PRP concentrate comprises the steps of preparing PRP from patient blood by capturing patient blood erythrocytes in a depth filter and preparing PRP concentrate by absorbing water in the PRP with absorbent beads. The method includes capturing the erythrocytes by rotating blood at centrifugal speeds in a balanced cylindrical separation chamber that is lined with the depth filter, the separation chamber and depth filter being segmented by radially extending plates into separation zones, the plates maintaining substantially balanced distribution of the blood in the separation zones during rotation of the separation chamber, thereby reducing vibration and erythrocyte displacement from the depth filter. 
     In this method, the rotational speed of the separation chamber can be accelerated to centrifugal speeds at a rate that allows balanced distribution of blood in the separation zones, and after the centrifuging is complete, the rotation speed of the separation chamber can be decelerated to below centrifugal speeds at a rate that allows balanced distribution of the PRP in the separation zones, thereby reducing vibration and erythrocyte displacement from the depth filter. The PRP can be contacted in a rotating concentrating chamber with desiccated beads to produce PRP concentrate while the beads are stirred with a stationary rake. The PRP concentrate can be collected by rotating the concentration chamber at centrifugal speeds to separate PRP concentrate from the beads. 
     The method for preparing PRP concentrate can comprise the steps of preparing PRP from patient blood by capturing patient blood erythrocytes in a depth filter, and preparing PRP concentrate by absorbing water in the PRP with absorbent beads. PRP concentrate can be produced by contacting PRP with desiccated beads in a rotating concentrating chamber while the beads are stirred with a stationary rake. The PRP concentrate can be collected by rotating the concentration chamber at centrifugal speeds to separate PRP concentrate from the beads. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a cross-sectional view of a disposable separation and concentration assembly and a permanent drive assembly, with desiccated beads shown in only half of the concentration subassembly. 
         FIG. 2  is a front view of the outer housing of the separation-concentration assembly of this invention. 
         FIG. 3  is a perspective view of the outer housing of  FIG. 2  showing details of the motor assembly connector. 
         FIG. 4  is a cross-sectional drawing of the separation-concentration sub-assemblies shown in  FIG. 1 . 
         FIG. 5  is a top view of the outer cap subassembly of the separation-concentration assembly shown in  FIG. 4 . 
         FIG. 6  is a cross-sectional view of the outer cap subassembly shown in  FIG. 5 , taken along the line  6 - 6 . 
         FIG. 7  is an exploded, isometric view of the outer cap subassembly shown in  FIG. 5 . 
         FIG. 8  is a top view of the top bucket cap subassembly of the separation-concentration assembly shown in  FIG. 4 . 
         FIG. 9  is a cross-sectional view of the top bucket cap subassembly shown in  FIG. 8 , taken along the line  9 - 9 . 
         FIG. 10  is an exploded view of the sample inlet subassembly. 
         FIG. 11  is a top view of the top bucket subassembly of the separation-concentration assembly shown in  FIG. 4 . 
         FIG. 12  is a cross-sectional view of the top bucket subassembly of  FIG. 11 , taken along the line  12 - 12 . 
         FIG. 13  is a front view of the valve assembly of the separation-concentration assembly shown in  FIG. 4 . 
         FIG. 14  is an exploded, isometric view of the valve assembly of  FIG. 13 . 
         FIG. 15  is a cross-sectional view of the bottom bucket subassembly shown in  FIG. 4 , taken along the central axis. 
         FIG. 16  is an enlarged cross-sectional view of the motor drive connector shown in  FIG. 15 . 
         FIG. 17  is a front view of the basket subassembly of the separation-concentration assembly shown in  FIG. 4 . 
         FIG. 18  is a cross-sectional view of the basket subassembly of  FIG. 16 , taken along the line  18 - 18 . 
         FIG. 19  is a top view of the mixer assembly of the separation-concentration assembly shown in  FIG. 4 . 
         FIG. 20  is a cross-sectional view of the mixer assembly of  FIG. 19 , taken along the line  20 - 20 . 
         FIG. 21  is an isometric view of the mixer assembly of  FIGS. 19 and 20 . 
         FIG. 22  is a perspective view of the motor drive assembly of this invention. 
         FIG. 23  is a cross-sectional view of the motor drive assembly of  FIG. 22  taken along the line  23 - 23 . 
         FIG. 24  is a cross-sectional view of the motor drive assembly of  FIG. 22  taken along the line  24 - 24 . 
         FIG. 25  is a cross-sectional view of the upper bucket and valve assembly of  FIG. 4 , taken along the central axis. 
         FIG. 26  is a cross-sectional view of the upper bucket and valve assembly of  FIG. 21 , taken along the line  26 - 26 . 
         FIG. 27  is a cross-sectional view of the upper bucket and valve assembly of  FIG. 4 , after the centrifugal action of the spinning upper bucket has extended the arms of the valve assembly and opened the valve. 
         FIG. 28  is a cross-sectional view of the view of upper bucket and valve assembly of  FIG. 27 , taken along the line  28 - 28 . 
         FIG. 29  is a cross-sectional view of the upper bucket and valve assembly of  FIG. 27 , after rotational displacement of the arms of the valve assembly. 
         FIG. 30  is a cross-sectional view of the upper bucket and valve assembly of  FIG. 29 , taken along the line  30 - 30 . 
         FIG. 31  is a cross-sectional view of the upper bucket and valve assembly of  FIG. 29 , after centrifugal separation has been completed. 
         FIG. 32  is a cross-sectional view of the separation and concentration assembly of  FIG. 1 , after blood has been introduced into the separation chamber. 
         FIG. 33  is a cross-sectional view of the separation and concentration assembly of  FIG. 32  as erythrocytes are separated from the plasma-platelet mixture during high speed centrifugation. 
         FIG. 34  is cross-sectional view of the separation and concentration assembly of  FIG. 33 , after platelet-plasma fraction has passed into the concentration chamber. 
         FIG. 35  is a cross-sectional view of the separation and concentration assembly of  FIG. 34  at the beginning of the high speed centrifugation to separate the platelet-plasma concentrate from the hydrogel bead. 
         FIG. 36  is a cross a cross-sectional view of the separation and concentration assembly of  FIG. 35  after platelet-plasma concentrate has collected in the platelet-plasma concentrate sump. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The apparatus and method of this invention prepares a novel PRP concentrate that combines enhanced platelet levels in a plasma concentrate in which the fibrinogen levels have not been significantly denatured. The novel product combines the sealant and haemostatic properties of the plasma concentrates greatly valued in certain types of surgery with the enhanced healing properties provided by elevated platelet levels. 
       FIG. 1  is a cross-sectional view of a disposable separation and concentration assembly and a permanent drive assembly, with desiccated beads shown in half of the concentration subassembly. Details of the sub-sections of this assembly are hereinafter described in conjunction with more detailed drawings. 
     The upper housing  2  is described in greater detail hereinbelow in conjunction with  FIGS. 2 and 3 . 
     The motor drive subsystem  4  is described together with the motor drive system in conjunction with  FIGS. 22-24 . 
     The separation system  3  enclosed in the upper housing  2  is described in greater detail with regard to  FIG. 4 . The separation system comprises a combination of subsystems including the outer cap subassembly  6  described in greater detail with respect to  FIGS. 5-7 ; a top bucket  8  described in greater detail with regard to  FIGS. 8 and 9 ; a sample inlet subassembly shown in  FIG. 10 ; a top bucket cap subassembly  10  described in greater detail with respect to  FIGS. 11 and 12 ; and a valve subassembly  12  described in greater detail with respect to  FIGS. 13 and 14 . 
     The concentrating system  11  includes a lower bucket  14  and drive connector  16 , described in greater detail with regard to  FIGS. 15 and 26 ; a basket subassembly  18  described in greater detail with regard to  FIGS. 17 and 18 ; and a mixer assembly described in greater detail with regard to  FIGS. 19 to 21 . 
       FIG. 2  is a front view of the outer housing of the separation-concentration assembly of this invention, and  FIG. 3  is a perspective view of the outer housing of  FIG. 2  showing details of the motor assembly connector. 
     The upper housing  2  isolates the sterile separation and concentration systems shown in  FIG. 1 . The upper portion of the outer housing  2  is sealed with an outer cap subassembly  34  having a blood inlet tube  86  and a PRP concentrate outlet port  62  and cap  66 . Referring to  FIG. 3 , the lower assembly connector has a drive recess  42  shaped to engage the motor subassembly, and with spacer receptors  44  for holding spacers  46 . The outer housing  2  and its enclosed separation components are a disposable unit to be used with a permanent drive assembly shown in  FIGS. 1 and 22  to  24 . The lower assembly includes an axially concentric motor drive receptor  48  and a plurality of tapered engagement and locking slots  50  that engage with corresponding mounting projections of the motor drive assembly (not shown). 
       FIG. 4  is a cross-sectional drawing of the separation-concentration sub-assemblies shown in  FIG. 1 . The outer housing  2  encloses an upper separation subassembly  3  and a lower concentration subassembly  11 . 
     The top of the outer housing  2  is closed with outer cap subassembly  6  shown in greater detail with regard to  FIGS. 5-7 . The outer cap subassembly  6  comprises a circular cap  56  with an annular flange  58  extending downward for securing it to the top of the upper housing  2 . Concentrate outlet conduit  60  passes through an outlet conduit hole  62  in the center of the plate  56 , extending through the plate and communicating with the separation chamber  64  ( FIG. 4 ). Circular cap  66  has a central receptor  68  that engages with a Luer fitting  70  on the upper end of the outlet conduit  60  to maintain a sterile closure during the separation process. 
     An inlet port hole  72  is positioned in the circular cap  56 , spaced from the central axis. The inlet port hole  72  is sized to engage the exterior inlet conduit  74  shown in  FIG. 4 . 
     The Luer fitting  70  is provided to engage an empty applicator syringe for removing platelet rich plasma concentrate product according to this invention. The lower end of the concentrate outlet conduit  60  constitutes a receptor for receiving the upper end of rigid tube  74  ( FIG. 4 ). 
     The bucket cap  10  shown in  FIG. 4  is described in greater detail with regard to  FIGS. 8 and 9 .  FIG. 8  is a top view of the top bucket cap subassembly  10  of the separation-concentration assembly shown in  FIG. 4 , and  FIG. 9  is a cross-sectional view of the top bucket cap subassembly shown in  FIG. 10 , taken along the line  9 - 9 . The cap subassembly  10  closes the top separation bucket  8  shown in greater detail with respect to  FIGS. 11 and 12 . The top bucket cap  10  comprises a circular plate  76  with a connecting flange  78  that extends downward from the lower edge of plate  76 . While the upper plate  6  is fixed to the outer housing  2  ( FIG. 4 ) and is stationary during the separation and concentration processes, top bucket cap  10  is secured to the top bucket  8  for rotation with the top bucket  8  during the separation and concentration processes. 
     The circular cap  10  has an axially concentric hole with a valve assembly guide tube  80  extending downwardly therefrom. The lower end of the guide tube  80  has a valve assembly stop flange  82  secured thereto. The upper end of the guide tube  80  supports sleeve bearing  84 . 
     The circular cap  10  has a sample inlet subassembly  86  that aligns with the hole  72  in the circular cap  56  ( FIG. 5 ). 
       FIG. 10  is an exploded view of the sample inlet subassembly  86 . The sample inlet subassembly  86  comprises an inlet tube  92  mounted in the plate  76 , the top of the inlet tube  92  including an annular receptor  94 . A sterile filter  96  is positioned in the lower end of the passageway  97  of tube  92 . 
     The subassembly  86  includes a removable inlet tube  98 . Inlet tube  98  comprises a central tube  100  having at its upper end an integral Luer fitting  102 . At an intermediate level of the tube  100 , an annular plate  103  extends outward from the tube. An integral cylindrical flange  104  extends downward from the outer edge of the plate  103 . The flange  104  is sized to engage the receptor  94 . The lower end  105  of the tube  100  is sized to engage the upper end of the passageway  97 . 
     The inlet tube is provided with a cap  106  that engages the Luer fitting  102  to provide a sterile closure of the removable inlet tube  98  during shipment and handling prior to use. 
     The inlet tube  98  in passing through the hole  72  in the stationary circular cap  56  locks the separation and concentration subassemblies against rotation during shipment and storage. After the patient blood is introduced into the top bucket  8  ( FIG. 4 ) through the inlet subassembly  86 , the inlet tube  98  is removed, unlocking the separation and concentration sub-assemblies  3  and  11  from the stationary circular cap  6 , freeing them for rotation about the central tube  74 . 
     A sterile breathing tube  108  is secured to the circular plate  76  to permit air flow from the separation chamber  64  when blood is introduced and to permit air movement into the system when platelet-rich concentrate is removed from the concentrating system  11 , as described in greater detail hereinafter. Sterile air filter  110  in breathing tube  108  ( FIG. 9 ) prevents entrance of micro-organisms into the interior of the separation chamber, preserving sterility. 
     The top bucket subassembly in  FIG. 4  is shown in detail in  FIGS. 11 and 12 .  FIG. 11  is a top view of the top bucket subassembly  10  of the separation-concentration assembly shown in  FIG. 4 , and  FIG. 12  is a cross-sectional view of the top bucket subassembly of  FIG. 11 , taken along the line  12 - 12 . The top bucket subassembly  10  comprises a cylindrical outer wall  112  having a top edge  114  that is secured to the inner surface of the flange  58  of the upper bucket cap  10 . The lower end of the cylindrical outer wall  112  is closed with integral sloped floor plate  116  with a central passageway  118  that constitutes a central flow passageway for separated platelet-plasma. The inner wall surface of the passageway  118  constitutes a valve seat  119  for the valve assembly described in greater detail hereinafter with respect to  FIGS. 13 and 14 . Spaced from the central passageway  118  and secured to the floor plate  116  are vent columns  120  with filters  122  in their bottom. The columns  120  serve as vents allowing movement of air from the concentration subassembly into the separation chamber when liquid flows through downward through the central passage  118 , as is explained hereinafter. Filters  122  prevent escape of hydrogel beads from the basket subassembly  18  through the vent columns  120  during transport or handling of the device of this invention. Surrounding the central passageway  118  and secured to the upper surface of the tapered floor plate  116  are upwardly extending abutment plates  124 , each having an upper valve arm abutment surface  128 . 
     A plurality of radially inwardly extending separation plates  130  are secured to the inner surface of the cylindrical outer wall  112  and the sloped floor plate  116 . Each adjacent pair of these plates defines a separation zone  132 . The plates  130  must be evenly spaced around the cylindrical outer wall to provide a balanced subassembly. They can be in matched, opposed pairs, for example the three matched sets as shown in  FIG. 11 . The top edge  134  of each of the separation plates  130  is spaced at a distance below the top edge  114  to permit overflow of blood in order to achieve an even distribution of blood between each the separation zones  132  during the spin acceleration stages and during the spin deacceleration stages, thus maintaining balance and minimizing vibration of the rotating assembly. 
     The interior surface  136  of the cylindrical outer wall segments in each of the each separation zones  132  is lined with an open-cell foam segment or depth filter segment  138 . The foam segments  138  have pores and passageways sized to allow infiltration of erythrocytes into the foam and subsequent entrapment of erythrocytes during the high speed centrifugation of the separation stage. The pores and passageways are sized to retain entrapped erythrocytes thereafter when the spinning slows or stops and the erythrocyte-free platelet-plasma suspension flows downward through the opening  118 . 
       FIG. 13  is a front view of the valve assembly  12  of the separation-concentration assembly shown in  FIG. 4 , and  FIG. 14  is an exploded, isometric view of the valve assembly of  FIG. 13 . The valve assembly  12  comprises a central tube  140 , the lower end constituting a valve face  142 . The valve face  142  comprises an annular receptor  144  that receives and holds an O-ring  146 . The outermost surface of the O-ring  146  is sized to form a sealing engagement with the valve seat  119  (See  FIGS. 11 and 12 ). 
     The valve assembly  12  includes two opposed centrifugal arms  148  secured to the tube  140  above the valve face  142 . Each centrifugal arm  148  has a flexible portion  150  adjacent the tube  140  and a rigid arm portion  152 . The distal end of the rigid arm portion  152  includes a weight receptor  154  in which a weight  156  is secured to provide additional weight to the end of the rigid arm portion. Operation of the valve assembly is described hereinafter with respect to  FIGS. 25-31 . 
     The lower bucket  14  in  FIG. 4  is shown in detail in  FIGS. 15 and 16 . Referring to  FIG. 15 , the lower bucket  14  has a cylindrical sidewall  158  and a sloped bucket bottom  160 , the lower portion of which forms a platelet-plasma concentrate sump  162  in which concentrated platelet and plasma concentrate collects. A plurality of basket supports  164  extend upward from the top surface of the slopped bucket bottom  160 , the top surfaces  166  of which support a concentrating basket subassembly  18  described hereinafter with regard to  FIGS. 17 and 18 . 
     An axially concentric drive receptor  168  shown in detail in  FIG. 16  is secured to the bottom surface of the slopped bucket bottom  160 . The drive connector receptor  168  can have any configuration that will releasably couple with a suitably configured motor drive connector. In the configuration shown in  FIGS. 15 and 16 , the drive receptor  168  comprises an outer cylinder  170  and a plurality of ridges  172 , each ridge having a tapered leading engagement surface  174 , an abutment surface  176  and an upper plate  178 . The upper plate  178  transmits the torque from the drive motor (described hereinafter with respect to  FIGS. 22-24 ) to the lower bucket bottom  160  and from there to the concentrating and separating subassemblies, all of which are secured together to form a unitary rotatable assembly. 
       FIG. 17  is a front view of the basket subassembly  18  of the separation-concentration assembly shown in  FIG. 4 , and  FIG. 18  is a cross-sectional view of the basket subassembly of  FIG. 17 , taken along the line  18 - 18 . The basket subassembly  18  comprises a cylinder  180  secured to a circular floor plate  182 . A slip bearing  184  is positioned in the axial center of the circular plate  182  for engaging the rigid tube  74  ( FIG. 4 ). The cylinder  180  has an array of windows  186  around its circumference, each window closed with a fine screen  188  having a mesh size sufficiently small to prevent escape of hydrogel beads  19  ( FIGS. 1 and 4 ) from the basket during spinning. 
       FIG. 19  is a top view of the mixer assembly of the separation-concentration assembly shown in  FIG. 4 .  FIG. 20  is a cross-sectional view of the mixer assembly of  FIG. 18 , taken along the line  20 - 20 , and  FIG. 21  is an isometric view of the mixer assembly of  FIGS. 19 and 20 . The mixer assembly  20  comprises a rake  190  secured to stationary tube  74 . The upper end  192  of the stationary tube  74  is secured to the upper cap subassembly  34  to secure it against rotation. The lower end  194  of the stationary tube  74  is an inlet port for removal of platelet-plasma concentrate from the sump  162  ( FIG. 15 ). The rake  190  comprises a radially extending spine  196  from which integral rake elements  198  extend downward to an elevation short of the bottom plate  182  of the basket subassembly  18  as shown in  FIGS. 4 ,  17  and  18 . The spine  196  can have optional breakaway notches  200  adjacent its center. The notches  200  weaken the spine and direct fracture of the spine  196  at the location of the notches if the event that the pressure produced by contact by beads  19  with the rake elements  198  during the final centrifugal spin become excessive. 
     The stationary tube  74  extends through the sleeve bearing  184  of the basket subassembly  18  and through the sleeve bearing  84  of the top bucket cap, permitting free rotation of the separating and concentrating assemblies around the stationary tube. The stationary tube  74  is fixed to the outer cap subassembly  6  and the stationary outer housing  2 . 
       FIG. 4  is a comprehensive assemblage of the components shown in  FIGS. 5-20 . 
     Concentrating desiccated hydrogel beads  19  fill the lower half of the basket  18  (only one side is shown empty to enable unobstructed viewing of the windows  186  and screen  188  elements ( FIGS. 17 and 18 ). 
     The concentrating desiccated hydrogel beads  19  can be insoluble beads or disks that will absorb a substantial volume of water and low molecular weight solutes while excluding high molecular weight solutes and particulates and will not introduce undesirable contaminants into the plasma. They can be dextranomer or acrylamide beads that are commercially available (Debrisan from Pharmacia and BIO-GEL P™ from Bio-Rad Laboratories, respectively). Alternatively, other concentrators can be used, such as SEPHADEX™ moisture or water absorbents (available from Pharmacia), silica gel, zeolites, cross-linked agarose, etc., in the form of insoluble inert beads. 
       FIG. 4  in conjunction with subassembly  FIGS. 5-21  shows the assembly prior to use with the valve assembly  12  secured for shipment by the sleeve  80  into which the valve assembly tube  140  extends and the abutment flange  82  secured to the bottom of the sleeve  80 . The valve face  142  is shown in position against the seat  119 . This confines the beads to the basket  18  and prevents escape of beads into the upper separation chamber  64  if the device is inverted or shaken during transport or handling. 
     The assembly is secured against rotation around the rigid tube  74  by the position of the removable inlet tube  98  in the hole  72  of the stationary outer cap subassembly  6 . 
     The upper edge of the cylinder  180  of the basket assembly  18  is secured against the lower surface of the tapered bottom  116 , and the lower surface of the plate  182  is secured against the upper edge surfaces  166  ( FIG. 15 ) of  the supports  164 . 
     Thus assembled, the upper separation subassembly  3  and the lower concentration subassembly  11  rotate as a single unit around the fixed tube  74 . The upper separation subassembly is positioned on the central tube  74  by the slip bearing  84  through which the fixed tube  74  extends. The lower separation subassembly is positioned on the central tube  74  by the slip bearing  184  through which the fixed tube extends. The rake assembly  20  including the tube  74  remain stationary during rotation of the separation and concentration subassemblies  3  and  11  in the separation and concentration phases, to be described in greater detail hereinafter. 
       FIG. 22  is a perspective view of the motor drive assembly of this invention.  FIG. 23  is a cross-sectional view of the motor drive assembly taken along the line  23 - 23 , and  FIG. 24  is a cross-sectional view of the motor drive assembly taken along the line  24 - 24 . 
     The outer shell  202  of the motor housing  4  encloses the motor  218  and supports the control interface  204  and the power connector  206 . The separation-concentrating assemblies are supported on the raised annular support surface  208  surrounding the motor connector  210 . Motor connector  210  has a configuration that will releasably engage the drive receptor  168  ( FIG. 16 ). The bottom of the housing  22  is closed by support plate  212 . A control and power plate  214  for the system is supported by four support struts  216  attached to the underside of the housing shell  202 . Plate  214  is a conventional printed circuit or equivalent board with the electronic components of the control and power system for the device, and in its center, a support  217  for the motor  218 . The electrical components are connected to the control interface  204  and power connector  206  by conventional wiring circuits (not shown). Four support feet  220  are secured to the bottom of the support plate  212  and provide friction surfaces  222  to secure the device on a laboratory surface. 
       FIGS. 25-31  illustrate the operation of the valve subassembly during and immediately after the initial separation process. Blood and blood products are omitted from these cross-sectional views to allow an unobstructed view of the valve assembly elements at each stage. 
       FIG. 25  is a cross-sectional view of the upper bucket and valve subassembly of  FIG. 4 , taken along the central axis, and  FIG. 26  is a cross-sectional view of the upper bucket and valve assembly of  FIG. 25 , taken along the line  26 - 26 . This is the view when blood is initially introduced into the top bucket  8 . The arms  148  of the valve subassembly are in their initial upright position, with the central tube  140  positioned in the guide tube  80  and the upper end of each arm contacting the flange  82 . The valve face  142  is in position in the valve seat  119  ( FIG. 12 ) at the upper end of the central passageway  118 , closing the passageway and preventing escape of blood. The flexible portions  150  of the arms  148  are positioned in the channels between the abutment plates  124  and  126 , preventing rotation of the arms  148  about tube  74  during shipment and handling. 
       FIG. 27  is a cross-sectional view of the upper bucket and valve assembly of  FIGS. 25 and 26 , after the centrifugal action of the spinning upper bucket has extended the arms of the valve assembly and opened the valve, and  FIG. 28  is a cross-sectional view of the view of upper bucket and valve assembly of  FIG. 27 , taken along the line  28 - 28 . After the desired volume of patient blood has been introduced into the top bucket  8 , the separation and concentration assembly is rotated around the tube  74  at a high speed, the centrifugal force created by this rotation causing the blood to flow outward and be distributed evenly by the separation plates into the separation zones  132 . The centrifugal force pools the blood against the outer surface of the foam segments  138  where the more dense erythrocytes preferentially move into the foam, leaving behind erythrocyte-free plasma containing the less dense platelets. 
     Under the force of centrifugation, the valve arms  148  rotate outward until they contact the sloped floor  116 . This action slides the valve central tube  140  upward to the upper portion of the guide cylinder  180 , pulling the valve face  142  from the central passageway  118  and out of contact with the valve seat  119  to open the passageway  118 . As the arms  148  rotate outward and the valve face  142  is lifted, the lower flexible ends  150  of the arms  148  are also pivoted upward from between the abutment plates  124  and  126 , freeing the arms for rotation about the tube  74 . Because the liquid is held against the foam segments  138  by centrifugal force, it does not flow through the open passageway  118 . 
       FIG. 29  is a cross-sectional view of the upper bucket and valve assembly of  FIGS. 27 and 28 , after rotational displacement of the arms of the valve assembly, and  FIG. 30  is a cross-sectional view of the valve structure of  FIG. 29 , taken along the line  30 - 30 . When the arms  148  are lifted from between the abutment plates and are freed from constraint by the abutment plates  124 , rotational motion causes the arms  148  to rotate about the rigid tube  74 . The rotation continues until one of the arms  148  contacts an adjacent separation plate  130  in its rotational path. This rotational displacement aligns the lower flexible ends  150  of the arms  148  above a portion of an abutment surface  128  of an abutment plate  124 . 
       FIG. 31  is a cross-sectional view of the upper bucket and valve assembly of  FIGS. 29 and 30 , after centrifugal separation has been completed and the rotation of the separation and concentration subassemblies is slowed or stopped. Under the force of gravity, the platelet-plasma mixture flows to the bottom of the tapered floor  116 , down its sloped surface to the central passageway  118 , and through the central passageway  118  to the basket subassembly  18  for concentration. The removal of the strong centrifugal action may permit the arms  148  to spring upward, causing the valve face  142  to move downward toward the central passageway  118 . This movement is stopped when one or both flexible arm portions  150  contact an opposed abutment surface  128 , leaving the central passageway open to the flow of the platelet-plasma mixture. 
     The operation of the device of this invention including the separation phase and concentrating phase are described hereinafter in conjunction with  FIGS. 32-36 . 
       FIG. 32  is a cross-sectional view of the separation and concentration assemblies of  FIG. 4 , after blood  202  has been introduced into the separation assembly  3  through the tube  110  from a syringe secured to the Luer fitting  102 . The upper tube  100  with the Luer fitting is then removed, unlocking the separation and concentration assemblies  3  and  11  for rotation. The blood flows into the bottom of the top bucket  8 . Air displaced by the incoming liquid escapes through breathing tube  108 . The valve face  142  is in a closed position, preventing escape of the blood from the bucket  8 . The operation of the system is then initiated, and the motor  218  spins the separation and concentration assemblies together around the rigid tube  74 . 
       FIG. 33  is a cross-sectional view of the separation and concentration assemblies of  FIG. 32  as erythrocytes are separated from the plasma-platelet mixture during high speed centrifugation. As the separation and concentration assemblies turn at a high speed, the blood is forced against the foam  138 . The erythrocytes, being more dense than other blood components, preferentially migrate into the pores and passageways of the foam. The valve subassembly opens the valve  142  as the centrifugal forces pivot the outer ends of the arms  148  away from the center, raising the valve face  142  face from valve seat  119  in the central passageway  118 . However, as long as the high speed centrifugation continues, all of the liquid is maintained against the foam. The centrifugal forces also force the hydrogel beads  19  radially outward against the outer screens  188  of the basket subassembly, out of contact with elements of the rake  190 . Centrifugation is continued until a majority of the erythrocytes a completely trapped in the foam. Because any erythrocytes weaken the gel product formed when the product of this invention is applied, the removal of a maximum proportion of the erythrocytes is desired. The speed of centrifugation tends to separate erythrocytes from platelets, leaving a substantial portion of the platelets in the plasma while entrapping a majority of the erythrocytes in the foam. 
       FIG. 34  is a cross-sectional view of the separation and concentration assembly of  FIG. 33 . After the spinning is slowed or stopped, the platelet-plasma fraction  204  flows to the bottom of the upper bucket  8  and down through the central passageway  118  into the basket subassembly  18  where it comes into contact with the desiccated hydrogel beads  19 . These beads concentrate the plasma by absorbing water from the liquid. The separation and concentrating assemblies are then rotated at a slow speed by the motor  218 , stirring the beads by moving them through the stationary spines  196  of the rake  190 . Agitating the beads insures maximum contact of the beads surfaces with the plasma and reduces gel polarization that arises when the plasma thickens adjacent the bead surfaces. This phase is continued until the desired proportion of the water has been removed and the desired concentration of the plasma has been achieved. 
       FIG. 35  is a cross-sectional view of the separation and concentration assembly of  FIG. 34  at the beginning of high speed centrifugation separation of the platelet-plasma concentrate from the hydrogel beads. At this stage, removal and maximum recovery of the platelet rich plasma concentrate  206  from the beads  19  is obtained. The separation and concentration assemblies are rapidly rotated by the motor  218  around the stationary tube  74 , creating centrifugal forces that force the platelet rich plasma concentrate and the beads  19  against the screen elements  188  of the basket  18 . The screen elements prevent escape of the beads  19  as the continuing centrifugal force causes the platelet enriched plasma concentrate to flow from the beads and through the screen. This high speed centrifugation is continued until a maximum recovery of the platelet rich plasma is obtained. 
     The absorption of water by the hydrogel beads is accompanied by an increase in bead diameter, increasing the bead volume. If the increased bead volume causes the ends of the rake  190  to drag on beads packed on the screen surface, the rake breaks along the break-away notches  200  ( FIG. 19 ), and the rake fragments become mixed with the beads. 
       FIG. 36  is a cross a cross-sectional view of the separation and concentration assembly of  FIG. 35  after the high speed centrifugation has ended and the platelet-plasma concentrate has flowed into the platelet-plasma concentrate sump  162 . The cap  66  has been removed, exposing Luer fitting  70  at the upper end of the tube  60 . An applicator syringe (not shown) is secured to the Luer fitting  70 . The platelet-rich plasma concentrate is removed from the sump  162  by retracting the barrel of the applicator syringe, drawing platelet rich plasma concentrate up through the tubes  74  and  60  and into the syringe. Breathing tube  108  permits air to flow into the system to replace the volume of liquid removed by the syringe, thus preventing the creation of a partial vacuum in the system that would impede liquid removal. 
     Regarding the concentration factor, for maximum wound-healing, the platelet level is maximized and high concentrate ion factors are sought. For homeostasis, plasma concentrations of 3 to 4 fold over anti-coagulated plasma levels are most effective. Concentrations below 3 fold have an insufficient fibrinogen concentrate ion. Concentrations higher than 4 old have excessive levels of total protein (principally albumin) which interferes with the fibrin gel structure. To obtain a preparation that maximizes haemostatic effectiveness while also providing improved (albeit perhaps less than maximal) wound-healing potential, a concentration range of 3 to 4 fold over anti-coagulated plasma levels is a best choice. For applications where sealant activity is not desired, high concentrations may be preferred. 
     Regarding erythrocyte levels, normal human hematocrits vary from 37 percent or lower to about 52 percent for whole blood, measured after a very high speed spin. To achieve concentrations of 3 fold or higher, some erythrocyte removal is necessary. However, the tensile strength of concentrated plasma gels diminish as the level of erythrocyte contamination increases. The concentration of erythrocytes in the final concentrate should be less than 3 to 5 percent to provide effective haemostatic properties. The device of this invention is intended to remove as much of the erythrocytes as is technically practical with the system, although trace contamination is accept able. For applications where sealant activities are not desired, higher levels of erythrocytes are tolerable. 
     Regarding volume, both the depth filter and the beads reduce the liquid volumes being processed. Because of this volume loss, only from 14 to 17 percent volume yields of effective haemostatic wound-healing product is generally obtained from average patient blood with the device of this invention. To make an effective product, the depth filter volume is selected to retain about 50 percent of the anti-coagulated blood (blood containing anticoagulant) and product about a 50 percent yield of PRP. The amount of the beads, in water absorption units, is selected to retain water equaling about 67 percent of the PRP volume. 
     Regarding accuracy, the amount of the depth filter and beads in each system is carefully selected to yield an optimum product. However, because of the wide range of hematocrit levels in patient populations, an approximate balance of components is required. 
     If too much blood is added to the device, there is a greater chance that the product will have a substantial erythrocyte contamination, and the final product will be less concentrated than desired because the volume exceeds the practical capacity of the depth filter. Because the volume retained by the depth filter is about half the total volume of blood to be processed, if the volume of blood introduced into the device is too small, a substantially lower volume of PRP will be delivered to the beads. For example, if the blood volume is low by only 25 percent, this will result in only 50 percent of the desired volume being delivered to the beads. If the volume of PRP contacting the beads is low by 33 percent or more, no product will be recovered because the beads will always absorb 67 percent of the targeted PRP volume. If the volume contacting the beads is only short by 17 percent, this will yield half of the desired volume of final product with twice the desired concentration (and hence of little value as a hemostat). In other words, a small error in the volume of blood introduced into the device is amplified into a large error in final product volume and concentration factor. 
     The systems can be designed to specifically match the hematocrit levels of the particular patient&#39;s blood to be processed. For a single optimized universal device, the device is optimized for the average patient blood, using fixed volumes of depth filter and blood, and a fixed bead water absorption capacity. 
     If it is desirable to tolerate inaccuracy of introduced blood volume, the device can incorporate an overflow chamber as described in provisional patent application Ser. No. 60/654,718 filed Feb. 17, 2005 and concurrently filed application Ser. No. 11/342,761, now U.S. Pat. No. 7,708,152, issued on May 4, 2010, the contents of which are hereby incorporated by reference. 
     EXAMPLE 
     Standard System Operation 
     Blood was processed with a device as shown and described in this application.
     1) The initial spin was continued for 10 seconds at 250 rpm. This spin allows beads to be flung out into the cage under sufficiently low rpm that the initial imbalance does not generate excessive vibration. The outer ends of the rakes (the outermost tines) level the beads around the perimeter of the basket to balance the beads.   2) The erythrocytes were separated with the an erythrocyte separation spin of 3200 rpm for 90 seconds, packing the erythrocytes into the depth filter.   3) The PRP was concentrated by slowing the spin to 50 rpm for 45 seconds, draining PRP into the concentrator chamber and mixing the PRP with the beads.   4) The PRP concentrate was then removed from the beads by a final high-speed spin at 3200 rpm for 45 seconds.
 
The rates of acceleration and deceleration between stages were moderated to reduce vibration.
   

     The process parameters were as follows: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Start Volume 
                 150 cc 
               
               
                   
                 Retained by depth filter 
                  75 cc 
               
               
                   
                 Recovered concentrate 
                  23 cc 
               
               
                   
                 Platelet count 
                 3 fold increase over whole blood 
               
               
                   
                 Fibrinogen concentration 
                 2.8-3.2 fold increase over while blood 
               
               
                   
                 Erythrocytes in product 
                 Undetected (less than 1%)

Technology Classification (CPC): 0