Patent Abstract:
An apparatus and method for collecting whole blood and then separating it into components for subsequent use or storage. A self-contained bag set is used to collect the sample, which may then be placed into a device adapted to fit into a centrifuge for separation of components. Each component is then sequentially extracted according to density, with a sensor present in the device to control the operation of valves directing the collection of each component. The sensor may be reading one or more of the following characteristics: infrared, optics, density, weight, radioactive, fluorescence, color, magnetism, ultrasonic, capacitance wherein the characteristic is inherent in the blood and blood component or is an additive. Each component may then be separated into its own storage container. The preferred sensors include optics and weight. Besides blood density separation, the device may contain a solution including cells, proteins, subcellular particles or viruses which may be mixed with affinity media or antibodies prior to separation.

Full Description:
FIELD OF THE INVENTION 
       [0001]    The following invention relates generally to instrumentalities and methodologies in blood component separation. More specifically, the instant invention is directed to a method and apparatus for collecting a blood sample and subsequently separating the collected sample into constituent blood components for individual storage or use. 
       BACKGROUND OF THE INVENTION 
       [0002]    Blood collection is always important, particularly in times of emergency (immediate use), but whole blood may only be stored for about 30 days before it is “outdated”. For long term storage, the ability to separate the whole blood into its major components (white blood cells, platelets, red blood cells and plasma) is of paramount importance because the long term storage condition for each component is different in terms of temperature and storage media. The most important component separations occurring after collection is the separation of red blood cells (RBC), white blood cells (WBC), platelets, and plasma from one another. Within the WBC it is sometimes important to separate the granulocytes from the lymphocytes and monocytes. After separation and extraction of particular components, a fraction of the blood may be returned to the patient. 
         [0003]    It is possible to separate the various components of whole blood either under or after centrifugation, due to their differing densities. Some prior art methods, such as that in U.S. Pat. No. 4,120,448, utilize a chamber connected to a centrifuge. The centrifuged blood separates in the chamber, and a plurality of collection means are positioned at various locations in the chamber corresponding to the areas where each component congregates, which is density-dependent. 
         [0004]    The present (prior art) technique for sequestering white blood cells from whole blood: requires skilled technicians, is labor intensive in that it requires 16 steps conducted over the span of one hour, and produces inconsistent results because of the requirements placed on the technician in the exercise of technique. Most significantly, however, the 16 step present technique is “open”; that is, the blood product is processed in a manner that does not maintain the sterility of the product because the need to obtain samples or add sedimenting agents or cryoprotectants at the various stages of production can not be accomplished with allowing the outside environment access to the interior, meaning potential contamination of the product: 
         [0005]    The 16 steps are: 
         [0006]    1. Collect placental blood into collection bag (range 60-200 ml). 
         [0007]    2. Add HES to collection bag (20% v/v). 
         [0008]    3. Load collection bag into special centrifuge cup supports. 
         [0009]    4. Centrifuge at 50 G for 13 min. to raise WBC from RBC (up to 6 units at one time). 
         [0010]    5. Spike or sterile dock collection bag to expressor and processing bag set to scale. 
         [0011]    6. Gently transfer collection bag to expressor and processing bag set to scale. 
         [0012]    7. Express off WBC rich plasma and 10-15 ml of the top layer of RBC into processing bag—leaving excess RBC. 
         [0013]    8. Remove collection bag with excess RBC. 
         [0014]    9. Load processing bag set in special centrifuge cup supports. 
         [0015]    10. Centrifuge processing bag set at 400 G for 10 min. (up to 6 units at one time). 
         [0016]    11. Gently transfer processing bag to expressor. 
         [0017]    12. Express off excess plasma leaving 20 ml WBC concentrate. 
         [0018]    13. Remove excess plasma bag from processing set. 
         [0019]    14. Add 5 ml cryoprotectant to WBC in processing bag at 4° C. 
         [0020]    15. Transfer cryoprotected WBC to freezing bag. 
         [0021]    16. Tube seal and separate freezing bag from processing bag. 
         [0022]    The following prior art reflects the state of the art of which applicant is aware and is included herewith to discharge applicant&#39;s acknowledged duty to disclose relevant prior art. It is stipulated, however, that none of these references teach singly nor render obvious when considered in any conceivable combination the nexus of the instant invention as disclosed in greater detail hereinafter and as particularly claimed. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 PATENT NO. 
                 ISSUE DATE 
                 INVENTOR 
               
               
                   
                   
               
             
             
               
                   
                 4,120,448 
                 Oct. 17, 1978 
                 Cullis 
               
               
                   
                 4,720,284 
                 Jan. 19, 1988 
                 McCarty 
               
               
                   
                 Des. 314,824 
                 Feb. 19, 1991 
                 Moon 
               
               
                   
                 5,674,173 
                 Oct. 7, 1997 
                 Hlavinka et al. 
               
               
                   
                 5,723,050 
                 Mar. 3, 1998 
                 Unger et al. 
               
               
                   
                 5,792,038 
                 Aug. 11, 1998 
                 Hlavinka 
               
               
                   
                 5,921,950 
                 Jul. 13, 1999 
                 Toavs et al. 
               
               
                   
                 6,315,706 
                 Nov. 13, 2001 
                 Unger et al. 
               
               
                   
                 6,348,031 
                 Feb. 19, 2002 
                 Unger et al. 
               
               
                   
                 6,652,475 
                 Nov. 25, 2003 
                 Sahines et al. 
               
               
                   
                 WO95/01842 
                 Published: Jan. 15, 1995 
                 Unger 
               
               
                   
                   
               
             
          
         
       
     
         [0023]    The prior art references listed above but not specifically described teach other devices for blood processing and further catalog the prior art of which the applicant is aware. These references diverge even more starkly from the reference specifically distinguished above. 
       SUMMARY OF THE INVENTION 
       [0024]    The present invention comprises a bag set that may be used to collect a whole blood sample from a source. Most significantly, the bag set defines a closed system in that once the blood is introduced, processing can occur outside a clean room or away from a sterile hood because access to any pathogens in the exterior environment is prevented. The bag set is then placed into a centrifuge for component separation. The whole blood processing bag, which may contain an anticoagulant such as CPD, ACD or CPD-A, contains at least one inlet and one outlet port connected to a plurality of component bags. The processing bag may optionally contain a sedimenting aid such as HES, but, unlike the prior art, such sedimenting aid is not required. Each component bag has a separate line leading from the whole blood processing bag, and each line can be clamped, tube-sealed and separated from the whole blood processing bag once a particular component bag has been filled. 
         [0025]    In practice, the blood is collected and directed into an inlet port on the whole blood processing bag and the input line is clamped, sealed off, and separated from the whole blood processing bag. The whole blood processing bag, which is asymmetrically shaped, hangs in a bag set holder having a complementally shaped opening that closely contacts the bag at the bottom end, and an exterior of the bag set holder is adapted to fit in a conventional centrifuge cup or socket. The centrifuge is operated at varying G-forces to optimally separate the components. Once the components are separated by density in the whole blood processing bag, a servo motor is engaged to open a metering valve on the line leading from the processing bag to a bag that will contain the densest component. This allows the densest component to fill its particular storage bag, usually under centrifugation. 
         [0026]    Applicant&#39;s process can be summarized in the following 7 or 8 steps which are performed over a span of 25 minutes, resulting in repeatable yields in excess of 90% of the lymphocytes and monocytes. 
         [0027]    1. Collect placental blood into collection bag (range 60-200 ml). 
         [0028]    2. Spike or sterile dock collection bag to bag processing set and transfer blood to processing bag. 
         [0029]    3. Add HES to processing bag (20% v/v). (Optional) 
         [0030]    4. Load processing bag set onto auto expresser. 
         [0031]    5. Centrifuge at an uninterrupted Run at two different speeds: 1,400 G for 20 min. to segregate WBC at RBC/plasma interface and 85 G for 5 min. to express the RBC to the RBC bag and WBC to freezing bag. 
         [0032]    6. Tube seal and separate excess RBC and plasma bags from processing set. 
         [0033]    7. Add 5 ml cryoprotectant to WBC in freezing bag at 4° C. 
         [0034]    8. Tube seal and separate freezing bag from cryoprotectant line. 
         [0035]    Complete collection of the first component is indicated preferably by an optical sensor that is present in the bag set holder device. The servo motor, directed by the sensor, automatically closes the metering valve on the line, terminating collection of that particular component. The servo motor then further engages the metering valve to allow collection of the next component through a second output line connecting the metering valve and the second storage bag. The process may sequentially continue until all desired components are collected in separate storage bags: red blood cells, white blood cells (lymphocytes and granulocytes), platelets, and plasma. If so desired, multiple components, such as the white blood cells and the platelets can be directed to the same storage bag. The sensor may be other than optical. For example, the sensor may monitor changes in electrical characteristics inherent in differing densities, such as capacitance, viewing the fluid as a dielectric. Commercially available markers (e.g. monoclonal antibodies, polarized particles, magnetic density, or fluorescence markers, etc.) can be introduced into the blood and monitored. 
         [0036]    The bags receiving fluid components may also be supported for weighing both during centrifugation and when at rest. Accurate separation occurs. 
         [0037]    Once collected, each storage bag may be sealed off and separated from the whole blood processing bag. Any necessary preservatives or additives may be introduced through the collection lines before processing or storing. 
       OBJECTS OF THE INVENTION 
       [0038]    Accordingly, it is a primary object of the present invention to provide a new and novel device and method for separating the components of whole blood for subsequent storage or use. 
         [0039]    It is a further object of the present invention to provide a device and method as characterized above in which separation may be accomplished entirely by machine during a single uninterrupted centrifugation run without the considerable handling between multiple centrifugation runs typically practiced in a blood bank with conventional means of separating blood components. 
         [0040]    A further object is to precisely sequester red blood cells, plasma, platelets and white blood cells even separating within white blood cell populations. 
         [0041]    It is a further object of the present invention to provide a device and method as characterized above in which the separation apparatus is self-contained to simplify the operation. 
         [0042]    Viewed from a first vantage point, it is an object of the present invention to provide a device for sequestering components from whole blood, comprising, in combination: a bag set, said bag set including a first bag and plural other bags; a bag set holder, whereupon the first bag is contained within an interior portion of the bag set holder, and the plural other bags are located at an elevation lower than said first bag; and a centrifuge having at least two diametrically opposed receiving sockets, at least one socket dimensioned to receive the bag set holder. 
         [0043]    Viewed from a second vantage point, it is an object of the present invention to provide an apparatus for use with a conventional centrifuge and a blood processing bag set, comprising, in combination: a first pocket having an unenclosed top portion, the first pocket dimensioned to receive a blood processing bag; means to support the blood processing bag in the first pocket, the support means located adjacent the unenclosed top portion of the first pocket; a movable bottom portion below the first pocket, the movable bottom portion having an open position and a closed position; a hinged portion located along a long axis of the first pocket, the hinged portion opening to allow access to the first pocket when the movable bottom portion is in the open position; and a second pocket, wherein access to the second pocket is only possible when the movable bottom portion is in the open position. 
         [0044]    Viewed from a third vantage point, it is an object of the present invention to provide a method for separating components from whole blood, the steps including: preparing a blood processing bag set having a processing bag, at least one auxiliary bag, a sampling site adjacent the processing bag, and a sampling site adjacent each auxiliary bag; introducing whole blood into the processing bag; sampling the whole blood for later analysis; centrifuging the whole blood, wherein components are separated in the processing bag; directing each component into the at least one auxiliary bag of the blood processing bag set; removing a sample of each component for later analysis; and storing each component for later use. 
         [0045]    Viewed from a fourth vantage point, it is an object of the present invention to provide a bag set, comprising, in combination: a first bag having an inlet and an outlet; plural auxiliary bags, each auxiliary bag having at least one port for admitting or expelling contents of the auxiliary bags; conduit means leading from the first bag to each auxiliary bag; valve means on the conduit means, the valve means adjustable to allow selective access between the first bag and the plural auxiliary bags. 
         [0046]    These and other objects will be made manifest when considering the following detailed specification when taken in conjunction with the appended drawing figures. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0047]      FIG. 1  shows the bag set holder of the present invention in open position. 
           [0048]      FIG. 2  shows the bag set holder of the present invention in closed position 
           [0049]      FIG. 3  shows the bag set of the present invention. 
           [0050]      FIG. 4  shows the bag set in position in the bag holder in open position. 
           [0051]      FIG. 5  shows the bag set in position in the bag holder in closed position. 
           [0052]      FIG. 6  shows positioning of two bag holders in a conventional centrifuge. 
           [0053]      FIG. 7  shows the bag set in the bag set holder before component separation. 
           [0054]      FIGS. 8A ,  8 B,  8 C show the stages of harvesting components from the processing bag. 
           [0055]      FIG. 9  shows the bag set in the bag set holder after component separation. 
           [0056]      FIG. 10  shows the bag set after collection of a blood sample before components are separated. 
           [0057]      FIG. 10   a  depicts the same state as  FIG. 10 , but without the intermediate buffycoat bag. 
           [0058]      FIG. 11  shows the bag set after the red blood cell component is separated. 
           [0059]      FIG. 11   a  depicts the same state as  FIG. 10 , but without the intermediate buffycoat bag. 
           [0060]      FIG. 12  is a flowchart of the preferred process. 
           [0061]      FIG. 13  illustrates the separation of whole blood components in graphical form. 
           [0062]      FIGS. 14A ,  14 B,  14 C show the operating positions of the metering valve. 
           [0063]      FIG. 15  shows an alternative embodiment of the bag set. 
           [0064]      FIG. 16  shows the attachment of a collection bag to the bag set. 
           [0065]      FIG. 17  shows the operation of draining the contents of the collection bag into the processing bag of the bag set. 
           [0066]      FIG. 18  shows the disconnection of the connection bag and clot filter from the bag set. 
           [0067]      FIG. 19  depicts the process of filling the sampling pillow with blood from the processing bag. 
           [0068]      FIG. 20  shows the disconnection of the sampling pillow and its associated sampling port from the bag set. 
           [0069]      FIG. 21  depicts the addition of an optional sedimenting agent to the processing bag. 
           [0070]      FIG. 22  illustrates the insertion of the bag set into the bag set holder. 
           [0071]      FIG. 23  is a depiction of the transfer of blood components that occurs under centrifuge while the bag set is in the bag set holder. 
           [0072]      FIG. 24  shows the disconnection of the red blood cell bag from the bag set. 
           [0073]      FIG. 25  illustrates the manner in which the contents of the freezing bag are mixed. 
           [0074]      FIG. 26  depicts the process of filling the sampling pigtail with the contents of the freezing bag. 
           [0075]      FIG. 27  shows the disconnection of the sampling pigtail and its associated sampling port from the bag set. 
           [0076]      FIG. 28  depicts the addition of DMSO into the freezer bag and its subsequent mixing. 
           [0077]      FIG. 29  illustrates the manner in which residual DMSO and air is drawn out of the system. 
           [0078]      FIG. 30  shows the disconnection of the freezing bag from the bag set. 
           [0079]      FIG. 31  illustrates the manner in which samples from the freezing bag portion are created for preservation. 
           [0080]      FIG. 32  shows the extraction of processing bag material and the small amount of freezing bag material left in the tubing from  FIG. 31  for subsequent analysis. 
           [0081]      FIG. 33  shows the disconnection of the DMSO inlet line and its associated junctions from the processing bag. 
           [0082]      FIG. 34  illustrates the manner in which samples are taken from the processing bag for subsequent analysis. 
           [0083]      FIG. 35  is a schematic of the servo motor and valve system connections. 
           [0084]      FIG. 36  plots, as a function of time while centrifuging: mass and liquid levels monitored by sensors. Also shown is the series of on/off valve rotations causing incremental weight increases of the harvested WBC solution, resulting in a full WBC freezing bag. 
           [0085]      FIG. 37  is a further iteration of a bag set schematically showing freezer bag (white blood cell) weighing during centrifugation. 
           [0086]      FIG. 38  reflects an alternate processing device  50 . 
           [0087]      FIG. 39  is another view of  FIG. 38 . 
       
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0088]    Considering the drawings, wherein like reference numerals denote like parts throughout the various drawing figures, reference numeral  10  as shown in  FIG. 3  is directed to the bag set according to the present invention. 
         [0089]    In its essence, the bag set  10  includes a whole blood processing bag  2 , a red blood cell (RBC) bag  4  having a hanger  16 , and a freezing bag  6  for the collection and storage of white blood cells. The processing bag  2  is supplied through an inlet line  12 , either through a phlebotomy needle  8  ( FIG. 10 ) or by being spiked, or sterile docked, to another bag containing the anti-coagulated blood. The processing bag  2  has an asymmetric shape including a top edge  11   a , a short side edge  11   b , a long side edge  11   c , and a sloped bottom edge  11   d  between the side edges such that the bottom portion tapers to an asymmetric point  14 , which leads to an outlet  26 . 
         [0090]    Asymmetric processing bag allows concention of a monuclear cell fraction of a white cell population in a time frame that excludes 30-50% of the granulocyte white cells. Granulocytes have no role in the hematoprietic reconstition and, thus their deletion results in a more purified selection of white cells for transplant. 
         [0091]    Also, the asymmetric bag set allows this purification to take place without the need for a sedimenting agent—which is too viscous to sterilize through a filter—thus allowing the MNC to be concentrated in a “closed” sterile bag set as the DMSO can be made sterile by passage through a 0.2μ filter at the cryoprotectant inlet to the bag set. 
         [0092]    The outlet  26  directs output from the processing bag  2  into a three-way metering valve  20 . The operating positions of the metering valve  20  are shown in  FIGS. 14A-14C . Two supply lines  24   a ,  24   b  lead from the metering valve  20  to the RBC bag  4  and the freezing bag  6 , respectively. The supply lines  24   a ,  24   b  and the inlet line  12  may each be heat sealed and separated from the bag set  10 . All lines are equipped with line clamps  22  that may be closed to prevent fluid passage when desired. If other components are to be separated, the bag set  10  may include additional bags with a corresponding adjustment to the metering valve  20  to accommodate the additional bags. 
         [0093]    Various supply lines may also be present in the bag set  10 . For example, the freezing bag supply line  24   b  may have an inlet  16  for the introduction of cryoprotectant into the system. Such inlets may be equipped with filters  30  (see, e.g.,  FIG. 10 ), preferably 0.2μ filters, to, inter alia, prevent contamination from pathogens in the outside air and to allow venting of air from the freezing bag and tubing. An intermediate buffycoat bag  40  ( FIG. 10 ) may be present on the freezing bag supply line  24   b . The buffycoat bag  40  collects a separate white cell fraction, which includes platelets and white cells and includes some small volume of plasma or red blood cells.  FIGS. 10   a  and  11   a  show the bag set without the intermediate buffycoat bag  40 . 
         [0094]    Initially, the processing bag  2  is either filled with an anticoagulant, such as CPD (citrate, phosphate, and dextrose) and blood is drawn through a phlebotomy needle into the bag, or the inlet line is spiked or sterile docked to another bag containing anticoagulated blood. The metering valve  20  begins in the closed position ( FIG. 8A ). All clamps  22  are closed with the exception of the clamp  22  on the inlet line  12 . Blood, preferably peripheral, placental umbilical cord blood, or bone marrow is obtained from a source through the phlebotomy needle  8  or other appropriate inlet, which feeds into the processing bag  2  through the inlet line  12 . The inlet line  12  is then clamped, heat sealed, and separated from the bag set  10 . Optionally, HES may be introduced into the RBC bag  4  through an optional inlet either before or after blood collection. 
         [0095]    At this point, the bag set  10  is placed in a bag holder  50 , shown in FIGS.  1 , 2 . The bag holder  50  is somewhat cylindrical, having a substantially elliptical shape, having two rounded ends connected by substantially straight sides. The main compartment  70  has an elongated oval shape dimensioned to receive the processing bag  2 . The main compartment  70  is accessed by sliding down a bottom portion  162  of the bag holder  50  (along arrow Z), then opening a cover  72  about a hinge  71  (along arrow X) present at one of the rounded ends of the bag holder  50 . The processing bag  2  is oriented in the bag holder  50  such that the hinged cover  72  closes over the edge  11   c  coinciding with the point  14  leading to the metering valve  20 . The metering valve  20  is received in an orifice  74   a  located on the major portion of the bag holder  50 . A complimental orifice  74   b , located on the hinged cover  72 , receives the protruding end of the metering valve  20 . The hinged cover  72  will only close when the bottom portion  162  is in the closed position. When the bottom portion is closed, a notch  164  in the bottom portion  162  registers with a retaining tab  166  present on the main body of the bag holder  50 . 
         [0096]    Referring to  FIG. 1 , the bag holder  50  includes a bag hanger  76  having hooks  60  that engage the loops  28  on the processing bag  2 , maintaining the bag in position during the centrifuging process. The main compartment  70  of the bag holder  50  is shaped to receive the processing bag  2 , having a sidewall  156  that is complemental to the asymmetric shape of the processing bag  2 , which terminates in an outport  160  dimensioned to receive the asymmetric point  14  and the outlet  26  of the processing bag  2 . The sidewalls  156  cradle the processing bag  2  loosely around the middle and more tightly at the bottom (near the outlet  26 ). Closer tolerance near the bottom of bag  2  is desired to minimize disturbing the contents of the bag after sedimentation. Thus, the top of compartment  70  mirrors the exterior elliptical shape but tapers down to the outport  160  while maintaining bag edges  11   b , 11   c , 11   d  in supporting relationship. 
         [0097]    A notch  78  is present along one of the substantially straight sides of the bag holder  50 . The notch  78  receives the hanger  16  on the RBC bag  4 . The RBC bag  4  hangs along the outside of the bag holder  50  in a curved recess  80  leading to a lower support shelf  83  via transition  81 . The freezing bag  6  is cradled in a receptacle  82  located beneath the main compartment  70  of the bag holder  50 , accessed by sliding the bottom portion  162  down to open along arrow Z.  FIGS. 4 and 5  show the entire bag set  10  loaded in the bag set holder  50  before component separation occurs.  FIG. 37  shows a further iteration of a bag set showing schematically that the freezer bag is weighed during the separation process.  FIG. 38  shows the freezer bag has been encapsulated in a shell  501  which depends from platform  503  that supports, on its top side a control chip module  57  and on its bottom side the shell and freezer bag via a weighing load cell  505 . Shell  501  floats in an air space  508 , protected by “U” shaped bracket  509 . 
         [0098]    The metering valve  20  is connected to a motor driver  56  in the bag holder  50 . The servo motor  56  is connected to a software-controlled control chip module  57  powered by a rechargeable battery B. Module  57  may require temperature compensation due to heat generation during centrifugation. A port P is provided to utilize a battery charger C ( FIG. 35 ). The servo motor  56  controls the operation of the metering valve  20  while the bag set  10  is mounted in the bag holder  50 . One or more optical sensors  58  trigger the proper time for the servo motor  56  to close the metering valve  20  after each fraction is harvested. The sensor may be present at the position shown in  FIG. 1  or lower, closer to the outport  160  ( FIG. 8C ) adjacent the asymmetric point  14  of the processing bag  2 . Sensors  58 , for example may monitor all branches around valve  20  and the inlets of bags  4  and  6 . The sensor  58  shown is optical but can be based on density, weight, infrared, radioactivity, fluorescence, color, magnetism, ultrasonics, capacitance, wherein the characteristic measured may be an additive. 
         [0099]    The bag holder  50 , when closed, is adapted to fit into a centrifuge cup  66  dimensioned to reside within a conventional centrifuge  100 . Preferably, at least two bag set holders  50  are placed in diametrically opposed centrifuge cups  66 , as shown in  FIG. 6 , for balance. A bag set  10  in the centrifuge cup  66  may be subjected to more than one G-force in order to achieve the optimum stratification of components ( FIGS. 8A-8C ). The servo motor  56  then operates the metering valve  20  to open and allow access to supply line  24   a  for the harvest of red blood cells, at an optimum G-force, into bag  4 . The servo motor  56  closes the metering valve  20  when the optical sensor  58  indicates that the red blood cells are harvested (FIGS.  8 A, 8 B). The optical sensor  58  senses the boundary between the white cell fraction and the plasma fraction. 
         [0100]    The next fraction, which includes white cells and/or platelets, is then harvested from the processing bag  2 ; the servo motor  56  opens the metering valve  20  to allow access to supply line  24   b  ( FIG. 8C ) leading to bag  6  for the next harvest. As shown in  FIG. 9 , during the harvest (WBC) into the freezing bag  6 , air in the supply line adds to air already in the freezing bag  6 , producing an air bubble  70 , which is useful to assist the proper mixing of the WBC and/or platelets with the cryoprotectant. The servo motor  56  then closes the metering valve  20 , as shown in  FIG. 8A , and the centrifuge  100  is allowed to stop.  FIG. 9  shows the bag set  10  in the bag set holder  50  after component separation has taken place. 
         [0101]    The buffycoat bag  40 , if present, preferably has a 25 ml capacity. 20 ml of buffycoat is introduced into the buffycoat bag  40 , and 5 ml of DMSO solution is subsequently introduced. The buffycoat bag is placed between two cold strata and rotating or kneading of the buffycoat bag  40  in order to mix the cryoprotectant and WBC solution takes place. 
         [0102]    The bag holder  50  is removed from the centrifuge cup  66  and opened, and the bag set  10  is removed, with the servo motor  56  disconnected from the metering valve  20 . Each supply line  24   a , 24   b  is clamped, heat sealed, and removed from the processing bag  2 . Any additional bags may be similarly removed. 
         [0103]    After the supply line  24   b  connected to the freezing bag  6  is disconnected, a cryoprotectant may be introduced into the collected component in the freezing bag  6  through an inlet. The air bubble  70  in the freezing bag  6  allows the cryoprotectant to be thoroughly mixed with the collected component. After mixing, the air bubble  70  is expelled, perhaps through a filter-protected cryoprotectant inlet  16  ( FIG. 10 ). The component is then prepared for storage by heat-sealing the tubing and removing the bag  6  downstream of the cryoprotectant inlet  16 . 
         [0104]    Preferably, each line (the inlet line  12  and the supply lines  24   a , 24   b ) is oriented to allow access to a sampling site (e.g., site  18 ) near the collection or storage bags. Thus, a sample of the blood or fluid in the line may be taken without disturbing the bulk of the collected component. 
         [0105]      FIG. 13  depicts the separation of whole blood components as a function of time. Under centrifugation, each fraction stratifies in the processing bag  2  as a function of its density. The overlapping areas  175  ( FIG. 13 ) indicate the area in the separation along each strata line in the processing bag  2 . As centrifugation continues, the boundary of each fraction becomes more clearly defined; thus, the area  175  ( FIG. 13 ) decreases and each fraction is more completely harvested. Thus, the centrifugation strategy combines separation by density, the time involved for stratification, which differs with the exterior surface area and density of the various cells, centrifugal force, and boundary layer clarity. Decisions on harvesting will vary based on these tradeoffs as a function of the constituent of greatest value and its desired purity. 
         [0106]    Preferably, the stratification centrifugation occurs at an excess of 1000 Gs, preferably 1400 Gs, for approximately 20 minutes. The transfer centrifugation step occurs at less than 100 Gs, preferably 78 Gs, and stops subject to output from the optical sensor  58 . The right hand side of  FIG. 36  shows the white cell bag (Freezer bag  6 ) topped off in increments by throttling the valve  20  on and off in order to extract the WBC population. 
         [0107]    It is appreciated that while the instant invention is preferably used in the separation of blood components, the separation techniques and apparatus are suitable for separation of other fluids. The software programmed into the control chip module may cause the servo motor to open and close the valve many times, thereby throttling the valve during strata delivery. Also by varying time increments during a harvest procedure, precise cut-offs between the cell components can be achieved in order to reduce the mixing between cell types that may occur as a result of the “toroidal” (Coriolis) effect during removal of the blood component from processing bag  2  and may be modified for the separation of other fluids or to compensate for various hardware conditions, such as uneven centrifuge loading. 
         [0108]    Yet another embodiment of the bag set  210  is shown in  FIG. 15 . In its essence, the bag set  210  includes a whole blood processing bag  202 , a red blood cell (RBC) bag  204 , and a freezing bag  206 . The processing bag  202  is supplied through an inlet line  212  that terminates in a spike  208 . The processing bag  202  has an asymmetric shape including a top edge  211   a , a short side edge  211   b , a long side edge  211   c , and a sloped bottom edge  211   d  between the side edges such that the bottom portion tapers to an asymmetric point  214 , which leads to an outlet  226 . The outlet  226  directs output from the processing bag  202  into a stopcock valve  220 . Two supply lines  224   a , 224   b  lead from the stopcock valve  220  to the RBC bag  204  and the freezing bag  206 , respectively. The supply lines  224   a , 224   b  and the inlet line  212  may each be heat sealed and separated from the bag set  210 . All lines are equipped with line clamps  222  that may be closed to prevent fluid passage when desired. If other components are to be separated, the bag set  210  may include additional bags with a corresponding adjustment to the stopcock valve  220  to accommodate the additional bags. 
         [0109]    Initially, the blood of interest is collected in a collection bag  200  or similar container. The spike  208  is inserted into the collection bag  200 , and the blood is drained from the collection bag  200  into the processing bag  202  through the inlet line  212  (FIGS.  16 , 17 ). The inlet line  212  preferably has a clot filter  230 , through which the blood passes before it reaches the processing bag  202 . After the blood is transferred, the inlet line  212  is heat sealed and the collection bag  200  and clot filter  230  are removed ( FIG. 18 ). 
         [0110]    The inlet line  212  also preferably has a sampling port  232 , a sampling pillow  234 , and an access port  236  ( FIG. 19 ). After the collection bag  200  and clot filter  230  are moved from the inlet line  212 , the sampling pillow  234  is squeezed and released to fill the sampling pillow with blood. The inlet line  212  is then heat sealed and the sampling pillow  234  is removed, along with the sampling port  232  ( FIG. 20 ). The blood in the sampling pillow  234  may then be accessed through the sampling port  232  for separate assay. 
         [0111]    Unlike the prior art where a sedimentation agent is required, a sedimenting agent, such as hydroxyethyl starch (HES) may optionally be added to the processing bag  202  through the access port  236  on the inlet line  212  using syringe means  236   a  or similar delivery means, and the processing bag  202  is manipulated to thoroughly mix the agent with the blood ( FIG. 21 ). The bag set  210  is then placed into the bag holder  50  and used with a centrifuge, as detailed hereinabove, to separate the cells therewithin ( FIG. 22 ). The separated red blood cells are transferred into the RBC bag  204  and the WBC fraction is transferred to the freezing bag  206  during this operation. The bag set  210  is then removed from the bag holder  50  ( FIG. 23 ). Supply line  224   a  is then heat sealed and the RBC bag  204  is removed ( FIG. 24 ). The contents of the RBC bag are accessed through a sample port  238 . 
         [0112]    Referring to  FIG. 25 , supply line  224   b  is preferentially equipped with a first junction  260  connecting an auxiliary inlet line  240  terminating in an auxiliary port  242 . A second junction  262  is present on the auxiliary inlet line  240  itself to connect a branch line  244  that terminates in a bulb  246 . The branch line  244  also contains a sampling pigtail  248  and a sampling port  250 . After removal of the RBC bag  204 , the bulb  246  on the branch line  244  is squeezed to direct any residual plasma remaining in the supply line  224   b  into the freezing bag  206 . Clamp  222  on branch line  244  is then closed. The contents of the freezing bag  206  are then mixed, preferably by holding the freezing bag  206  at a 45° angle and slowly squeezing the small compartment  206   a  of the freezer bag  206  a total of ten times at one squeeze per second. 
         [0113]    The clamp  222  on the branch line  244  is then opened, and the bulb  246  is squeezed and released to fill the sampling pigtail  248  with the contents of the freezer bag  206  ( FIG. 26 ). The branch line  244  is heat sealed and removed from the bag set  210  ( FIG. 27 ). The contents of the sampling pigtail  248  are accessed through the sampling port  250  for separate assay. 
         [0114]    The freezing bag  206  is placed on its side and sandwiched between two ice packs  252  ( FIG. 28 ). DMSO is introduced into the freezing bag  206  through the auxiliary port  242  which has a sterile filter  242   a  (i.e. less than or equal to 0.2 microns) on the auxiliary inlet line  240 . An orbital mixer  254  is used with the sandwiched freezer bag  206  to thoroughly mix the contents of the freezer bag  206 . The sandwiched freezer bag  206  is then placed in stationary holder  256  ( FIG. 29 ). A syringe  258  is inserted into the auxiliary inlet  242  and used to draw out any residual DMSO and trapped air in the supply line  224   b  and the auxiliary inlet line  240 . The buffy coat/DMSO from the freezing bag  206  is drawn out by the syringe  258  until it reaches the second junction  262  from the supply line  224   b . The freezing bag  206  is then removed from the bag set  210  by heat sealing the supply line  224   b  ( FIG. 30 ). 
         [0115]    A portion of the supply line  224   b  after the first junction  260  remains attached to the freezing bag  206 . This portion of the supply line  224   b  is heat sealed to form three separate samples  275   a , 275   b , 275   c  (still connected to the freezing bag  206 ), and the area separating the small compartment  206   a  of the freezer bag  206  is heat sealed to separate it from the rest of the freezer bag  206  ( FIG. 31 ). The final product is then frozen for storage. 
         [0116]    The stopcock valve  220  is turned to allow plasma in the processing bag  202  to contact the buffy coat in the supply line  224   b  near the first and second junctions  260 , 262  ( FIG. 32 ). A sample of the plasma diluted buffy coat is drawn into the syringe  258  for bacterial sampling, and the syringe  258  is removed from the auxiliary port  242 . The supply line  224   b  containing the auxiliary line  240  and the first and second junctions  260 , 262  is then disconnected from the processing bag  202  and is discarded ( FIG. 33 ). Samples of the plasma in the processing bag  202  may be removed by using the access port  236  ( FIG. 34 ). 
         [0117]    Moreover, having thus described the invention, it should be apparent that numerous structural modifications and adaptations may be resorted to without departing from the scope and fair meaning of the instant invention as set forth hereinabove and as described hereinbelow by the claims.

Technology Classification (CPC): 0