Patent Publication Number: US-4482342-A

Title: Blood processing system for cell washing

Description:
DESCRIPTION 
     1. Technical Field This invention is in the field of fluid processing and more particularly relates to the centrifugal separation of fluid, such as blood, into two or more components, such as during cell washing or component separation. 
     2. Background Art It is often desirable to transfuse only the red blood cells to a transfusion recipient since red blood cells (RBC&#39;s) are not known to cause an immunological reaction in a recipient. 
     Present state of the art processes for initially separating donated whole blood into its component elements, such as RBC&#39;s, platelets and plasma proteins and white blood cells, are not sufficiently effective in entirely removing substantially all undesirable components from the RBC&#39;s. 
     It is therefore necessary to provide a system and procedure for &#34;washing&#34; the packed RBC&#39;s. (Note: The term &#34;packed RBC&#39;s&#34; will hereafter be used to refer to unwashed RBC&#39;s which have been separated from other whole blood components). The packed RBC&#39;s are washed with a wash solution, such as saline, to remove such undesirable components remaining after the initial centrifugal separation. Such undesirable components, unlike RBC&#39;s are known to cause adverse transfusion reactions. 
     The packed RBC&#39;s can be washed in a number of ways. One method now in practice is to centrifuge a unit of donor blood in a collection bag and subsequently remove the plasma and buffy coat manually using a plasma expressor leaving packed RBC&#39;s in the collection bag. Then the packed RBC&#39;s are diluted with saline, centrifuged again, and the supernatant manually removed using an expressor, leaving washed RBC&#39;s. 
     Packed RBC&#39;s can also be washed by diluting the packed RBC&#39;s with saline in a centrifugal processing bag or bowl and expressing the supernatant through a rotating seal leaving washed cells. The Haemonetics Model 102 cell washing equipment is of the bowl type. (See: The Preparation of Leukocyte-Poor Red Blood Cells: A Comparative Study Meryman et al., Transfusion 20(3):285:287,1980) 
     The IBM 2991 Cell Washer (generally described in U.S. Pat. Nos. 4,007,871 and 4,010,894) utilizes a spin and agitation method in which packed RBC&#39;s are spun within a saline solution in a toroidal chamber of fixed volume and then agitated in the chamber. This process is repeated many times with fresh wash solution until sufficient hematocrit of the washed RBC&#39;s is attained. The agitation is required in order to maximize interaction between the wash solution and the packed RBC&#39;s. The IBM 2991 is effective in washing but the apparatus is complex and thus expensive and the procedure very time comsuming. (See: Use and Analysis of Saline Washed Red Blood Cells Wooten, M. J., Transfusion 16(5):464 1976) 
     Accordingly, it would be desirable to provide washing apparatus and methods which are simple, inexpensive and speedy. 
     DESCRIPTION OF THE INVENTION 
     In the method and apparatus of the present invention, packed RBC&#39;s are washed by a suitable wash solution within a centrifugal processing bag. The efficiency of the cell washing procedure is optimized by initially orienting the processing bag with respect to the axis of the centrifuge center of rotation (CR), such that (1) the highest density component, i.e., washed RBC&#39;s, accumulate at a corner of the bag furthest from the axis of the CR and locating the inlet port for the wash solution at that corner and (2) the lowest density component i.e., supernatant accumulates at a corner of the bag which is closest to the axis of the CR and locating the outlet port at this latter corner. This orientation is accomplished by means of a &#34;double angled&#34; cassette support which forces the processing bag to assume a position in the centrifuge rotor which is at an angle with respect to the axis of rotation and also at an angle with respect to the position of concentricity; hence the term &#34;double angled&#34;. 
     The cassette support member is tilted inwardly from the vertical plane and the cylindrical segment shape of the member is off-set to be eccentric with the axis of the CR. The whole blood bag may be rectangularly shaped with four corners labelled A, B, C and D, counterclockwise from the upper right-hand corner &#34;A&#34; (looking from the CR). The bag is positioned adjacent the double angle support member. The tilted eccentric shape of the support member forces the bag to assume an orientation with respect to the axis of the CR such that: 
     
         r.sub.1 &lt;r.sub.2 &lt;r.sub.4 
    
     and 
     
         r.sub.1 &lt;r.sub.3 &lt;r.sub.4 wherein r.sub.1, r.sub.2, r.sub.4 and r.sub.3 are the radii from respective bag corners A, B, C and D to the axis of the CR. 
    
     The supernatant outlet port is located at the shortest radius r 1 , in this case, the upper right-hand corner &#34;A&#34;, and the wash solution input port is located at the longest radius r 4 , in this case, the lower left-hand corner &#34;C&#34;. With the outlet port at the shortest radius, the lower density supernatant component can be readily removed through this port. Furthermore, by introducing the wash solution at the longest radius port location the wash solution interacts with or &#34;sees&#34; the most packed RBC&#39;s. Also, a turbulent flow is created whereby the cells are agitated thereby maximizing the cell washing efficiency. 
     These and other advantages will become apparent from the following description of the best mode for carrying out the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a simplified top view of an embodiment of the invention. 
     FIG. 2 is a cross-sectional view through lines 2--2 of FIG. 1. 
     FIG. 3 is a plan view of a disposable software set utilized in an embodiment of the invention. 
     FIG. 4 is an enlarged plan view of the bag structure illustrating further details of the invention. 
     FIG. 5 is a schematic illustration of the controls for cell washing. 
     FIG. 6 is a schematic representation of the back support member 9 and bag 8 of the invention. 
     FIG. 7 is a perspective view of the double-angle back support member. 
     FIGS. 8A and 8B are a diagramatic sectional illustration of the details of the invention with a support 100 (FIG. 8A) and without a support (FIG. 8B). 
    
    
     BEST MODE FOR CARRYING OUT THE INVENTION GERNERAL PROCEDURE 
     In general, it may be seen that this invention comprises an apparatus and process for washing blood components, particularly packed RBC&#39;s, with a suitable wash solution, such as a saline solution. The invention is not, however, intended to be limited to cell washing and may find applicability in other areas, such as plasmapheresis or plateletpheresis. 
     The invention will be described in connection with a specific centrifuge apparatus found in certain copending applications. Because of the imbalances produced in the processes to be described, it is desirable that a Self-Balancing Centrifuge as described in U.S. patent application Ser. No. 281,648 filed 9 Jul. 1981 now U.S. Pat. No. 4.412,831, (hereby incorporated by reference), or equivalent, will supply the necessary centrifugal force for blood processing in accordance with the invention. However, the invention is not intended to be limited to any particular centrifuge. 
     Furthermore, the invention will be described in connection with a copending U.S. patent application Ser. No. 281,655 filed 9 July 1981 (hereby incorporated by reference), which describes a new and improved pheresis process and apparatus generally constructed as follows. A first container, in the form of a flexible bag containing anticoagulated whole blood to be centrifugally separated, is supported by a cassette located on a centrifuge rotor. The cassette is in the form of a rack or stand partitioned into three annular sections by two vertically positioned support members. Each member has a shape generally described by a segment of a cylinder with a radius of curvature corresponding to a radius to the axis of the center of rotation (CR) of the centrifuge rotor. A second container is disposed in the cassette adjacent the first container and in fluid communication with the first container. The second container, which may also be a flexible bag, is adapted to receive one or more of the centrifugally separated components of the anticoagulated whole blood. 
     A pressure plate in the form of a body of material such as a metal plate also having a curvature corresponding to a radius to the axis of the centrifuge CR, and having a predetermined mass is disposed between the first bag and the center of rotation of the rotor. This pressure plate is suspended so that it is free to move radially against the first bag when subjected to the centrifugal forces generated by rotation of the centrifuge. The pressure plate has a predetermined mass sufficient to at least initiate a flow of separated fluid component from the first bag to the second bag as the pressure plate presses against the first bag during rotation of the centrifuge rotor. The mass distribution and shape of the pressure plate is adapted to pool the separated first blood component in the area of an umbilical fitment on the bag. An output port is located at this fitment. 
     The first bag and second bag are located adjacent each other on the rotor with the first bag positioned radially inward from the second bag. A siphon effect is created when flow is initiated from the first bag to the second bag as the pressure plate pushes against the first bag under the influence of centrifugal force. The siphon effect is due to the difference in centrifugal forces to which the bags are subjected because one bag is located nearer the center of rotation than the other. 
     As will subsequently be described, the combination of the pressure plate flow initiation and siphon effect described in the above referenced U.S. patent application Ser. No. 281,655 will be used in the present application in connection with a cell washing procedure and is therefore hereby incorporated by reference. 
     Referring now to the apparatus of FIGS. 1 and 2, a double-angle cassette support member 9 (to be described in detail in connection with FIGS. 6 and 7) is located on one side of a centrifuge rotor 28 near the periphery. A weight plate 15 is located adjacent the cassette support member 9 and is free to move radially under the influence of centrifugal force toward the support member 9. A processing bag 8 is disposed between the weight plate 15 and the support member 9 and is oriented by support member 9 in a position such that lighter density component accumulates at the upper right-hand corner &#34;A&#34; of the bag 8 and heavier density component accumulates at the lower left-hand corner &#34;C&#34; of the bag 8. A deformable support member 100 is provided at corner &#34;A&#34; to insure that the outlet at port &#34;A&#34; is located sufficiently near the axis of the CR to enable all of the lighter density component (supernatant) to exit the port located at corner &#34;A&#34;. 
     The blood processing/cell washing bag 8 is in fluid communication with (1) the wash solution bag 20 via wash line 25, (2) a supernatant bag 2 via supernatant line 27, and (3) a packed RBC&#39;s bag 4 via fill line 23. Line 27 is coupled through a solenoid actuated clamp 92c. Wash line 25 is coupled through a second solenoid actuated clamp 92d. Each of these clamps are supported on vertical support members 74a and 74b, which along with vertical support member 74c form an H-shaped vertical support structure to which various fixed components of the cell washing process may be attached. Supernatant bag 2 is disposed at the periphery of the centrifuge rotor opposite the double-angled cassette support member 9. Wash solution bag 20 is located between a cassette support 11 and a weight plate 17 at a location nearer the axis of the CR of the rotor than the blood processing/cell washing bag 8, but on the opposite side of the axis of the CR from bag 8. The packed RBC&#39;s bag 4 is located between a RBC cassette support 13 and a weight plate 19 at a location nearer the axis of the CR of the rotor than the blood processing bag 8 and on the same side of the CR as bag 8. 
     Before cell washing; anticoagulated whole blood is centrifugally separated in whole blood bag 4 in a swinging bucket centrifuge (not shown). The plasma is then manually expressed leaving behind packed RBC&#39;s in bag 4. 
     To wash the packed RBC&#39;s, the bag 4 is placed in the RBC cassette between the support 13 and the weight plate 19. The packed RBC&#39;s bag 4 is connected by bag spike 43a (See FIG. 3) and conduit 23 to processing bag 8. Processing bag 8 30 is placed between the double angle cassette 9 and weight plate 15. Similarly, the wash solution bag 20 is attached to the processing bag 8 with bag spike 43b (See FIG. 3) and placed between cassette support 11 and weight plate 17. All of the weight plates and supports, with the exception of the support member 9 and weight plate 15, are similar to those described in the previously referenced application, Ser. No. 281,655. 
     The conduit from supernatant bag 2 is labelled 27 in FIG. 3, and as seen in FIGS. 1, 2 and 5 is disposed between optical sensor 90 and solenoid actuator clamp 92c. Similarly, the conduit 25 between the port 47 and the wash solution bag 20 is disposed between a solenoid actuated clamp 92d. The control circuitry for the clamps 92c and 92d is shown in FIG. 5. 
     Having made these connections, the apparatus is now ready for a cell washing procedure. The centrifuge is rotated, causing pressure plate 19 to press against packed RBC&#39;s bag 4 expressing the contents into the processing bag 8. At this point in time, conduit 27 has been clamped off by clamp 92c. Furthermore, initially, conduit 25 is clamped until sufficient dwell time is achieved to insure that the RBC&#39;s have accumulated at port 47. After this dwell time has elapsed, the clamp 92d on conduit 25 is opened, the wash solution is expressed into the processing bag 8, now containing packed RBC&#39;s. This is accomplished while the centrifuge is spinning and cell washing takes place, as shown generally in FIG. 4. 
     After a sufficient period of centrifugation has occurred, the washed RBC&#39;s will accumulate at the lower left-hand corner &#34;C&#34; of the processing bag 8, and supernatant will accumulate at the upper right-hand corner &#34;A&#34;. 
     Next, the conduit 27 connected to port 45 of the processing bag is unclamped by operation of clamp 92c to permit passage of supernatant from processing bag 8 to supernatant bag 2. A siphon effect is created when flow is initiated from the processing bag 8 to the supernatant bag 2 as the pressure plate 15 pushes against the processing bag under the influence of centrifugal force. The siphon effect is due to the difference in centrifugal forces to which the bags are subjected because one bag is located nearer the center of rotation than the other. 
     Optical sensor 90 senses when red blood cells pass through conduit 27 whereupon it provides a signal to control 94a which energizes clamp 92c to clamp conduit 27 and prevents further flow from the processing bag 8. 
     The procedure of expressing saline into the processing bag 8 through conduit 25 and removing supernatant from processing bag 8 through conduit 27 may be repeated several times to assure an optimal removal of plasma, platelets, white blood cells and cell debris from the packed RBC&#39;s. 
     After the wash procedure is completed, the centrifuge can be stopped, and the conduit 27 to the supernatant bag 2 may be manually clamped and severed from the processing bag which now contains the washed RBC&#39;s. Likewise, the packed RBC&#39;s bag 4 and the wash bag 20 may be severed from the processing bag 8 and the RBC&#39;s, which have now been centrifugally washed, may be reintroduced to a patient. 
     Double Angled Support Member 
     The construction of the processing bag 8 and its corresponding back support member 9 is unique to this invention and will be described in some detail in connection with FIGS. 6-8. 
     In the method and apparatus of the present invention, as shown in FIG. 6, the processing bag 8 is oriented by a rigid back support member 9, such that (1) the highest density component (RBC&#39;s) accumulate at the lower left-hand corner of the bag furthest from the axis of the center of rotation (CR) of the centrifuge and where the inlet port 47 is located and (2) the lowest density component (supernatant) and wash solution accumulate at the upper right-hand corner of the bag which is closest to the axis of the CR of the centrifuge and where the outlet port 45 is located, as shown in FIG. 7. This is accomplished by utilizing a &#34;double angled&#34; cassette support member 9, shown in FIG. 7, which orients the processing bag in the centrifuge rotor at an angle with respect to the axis of rotation and also at an angle with respect to the position of concentricity; hence the term &#34;double angled&#34;. To do this, the cassette support member 9 is tilted inwardly from the vertical plane and the cylindrical segment shape of the member 9 is formed eccentric with the axis of the CR. The degree of tilt is preferably sufficient to provide a maximum separation gradient consistent with the permissable space provided in the centrifuge rotor. Likewise, the degree of eccentricity of the support member is predicted on achieving good separation within the limitations of space. 
     The pressure plate (15 in FIG. 7) is a body of material such as a metal or plastic plate having a curvature generally corresponding to the curvature of the support member 9 and having a predetermined mass. The plate is disposed between the processing bag and the axis of the CR of the rotor. This pressure plate 9 is suspended so that it is free to move radially against the processing bag 8 when subjected to the centrifugal forces generated by rotation of the centrifuge. The pressure plate 9 has a predetermined mass sufficient to at least initiate a flow of separated fluid component from the processing bag 8 to the supernatant bag 2 as the pressure plate 9 presses against the processing bag 8 during rotation of the centrifuge rotor. The mass distribution and shape of the pressure plate 9 is adapted to pool the separated component in the area of the outlet port 45. 
     Bag 8 is rectangular in shape (as can be seen more clearly in FIG. 4 with four corners A, B, C, and D, lettered counterclockwise from the upper right-hand corner. Preferably, the bag is manufactured from two sheets of PVC welded together at the edges. In order to utilize inexpensive materials in the fabrication of such bags and yet withstand the pressures generated during separation, it may be desirable to utilize a support structure for each bag as described in copending U.S. patent application Ser. No. 339,910filed 18 Jan. 1982. 
     When the bag is positioned in the support member 9, the tilted eccentric shape of the support member forces the bag to assume an orientation, with respect to the axis of the CR (See FIG. 6) such that 
     
         r.sub.1 &lt;r.sub.2 &lt;r.sub.4 
    
     and 
     
         r.sub.1 &lt;r.sub.3 &lt;r.sub.4 
    
     wherein r 1 , r 2 , r 4  and r 3  are the radii from respective bag corners A, B, C and D to the axis of the CR. 
     The outlet port 45 to the supernatant bag 2 is located at the shortest radius r 1 , in this case, the upper right-hand corner &#34;A&#34;, and the wash solution input port 47 is located at the longest radius r 4 , in this case the lower left-hand corner &#34;C&#34;. With the outlet port at the shortest radius, the lower density component can be removed through this port. Furthermore, by introducing the wash solution at the longest radius port location, the solution &#34;sees&#34; the most packed RBC&#39;s and generates a counter-current flow through the red cells, thus maximizing cell washing efficiency. 
     Software Set 
     Referring now to FIG. 3, there is shown a software set suitable for use in connection with the present invention. The software set consists essentially of two fluid interconnected flexible bags 2 and 8, plus two accessory bags 4 and 20. Bags 4 and 20 are not initially interconnected with the other bags but bag 8 is equipped with conduits 23 and 25, respectively, at the end of which bag spikes 43b and 43a are provided to enable fluid communication with ports 42 on each bag for cell washing. 
     Bag 4 contains packed RBC&#39;s which are expressed into processing bag 8 via port 40 for cell washing with a wash solution from bag 20. The wash solution is expressed into corner port 47 from bag 20 via conduit 25. Supernatant from the wash procedure is expressed from bag 8 via outlet corner port 45 and conduit 27 to supernatant bag 2. 
     As previously noted in connection with bag 8, these bags are preferably made of suitable thin walled hemo-compatible plastic material, such as polyvinyl chloride (PVC). The basic construction of these bags consists of forming two sheets of material in accordance with the desired bag shape and welding the edges of the sheets together to form an interior chamber for the bag. 
     As may be seen clearly in FIG. 4, using the &#34;double angle&#34; orientation for cell washing allows the introduction of the saline at the location where it can &#34;see&#34; the most red cells. The more dense red cells pack at the outer-most radius, corner &#34;C&#34; in FIG. 4. Introducing saline from a port 47 in that corner directs the saline through the bed of packed cells. A counter-current flow is generated by centrifugal forces without the necessity for a separate agitation cycle. The less dense saline moves from corner &#34;C&#34; to corner &#34;A&#34;, while the red cells move in the opposite direction right back into the stream of saline at corner &#34;C&#34;. The saline carries other less dense components (supernatant) such as platelets, white blood cells and plasma proteins with it leaving only washed red cells in corner &#34;C&#34;. The supernatant, including the platelets, white blood cells and plasma proteins can then be expressed through port 45 at corner &#34;A&#34;. 
     Several details about the construction of processing bag 8 are important. The wash inlet port 47 should be small enough in diameter to provide a turbulent jet to promote mixing of the wash solution and RBC&#39;s. A 1/16 inch diameter inlet port with a 300 ml/minute saline wash solution flow rate provides acceptable results. In addition, we have found that the inlet port 47 at &#34;C&#34; works best if it is disposed at an angle of from 30° to 90° with respect to the side of the bag. This directs the saline into the center of where the RBC&#39;s have packed. It may also be desirable to create a more diagonal shaped bag as indicated by the dashed lines in FIG. 4. This would prevent packing in corners &#34;B&#34; and &#34;D&#34; where the wash solution jet may not reach the cells. 
     We have found through tests using a self-balancing centrifuge, as described in U.S. patent application Ser. No. 281,648, and a double-angled blood processing bag, as described herein, that the geometry of the bag is critical to the effective expression of wash solution and plasma (low-density component) from red blood cells (high-density component). In particular, the radius (measured from the axis of rotation to the processing bag) should consistently decrease from (See FIG. 6): 
     C to A : (r 4  to r 1 ) 
     C to D : (r 4  to r 3 ) 
     C to B : (r 4  to r 2 ) 
     D to A : (r 3  to r 1 ) 
     B to A : (r 2  to r 1 ) 
     If the radius does not consistently decrease, the higher density component will pack in the area of increasing radius. For example, referring to FIG. 6, in an experiment with blood, the radius r 4  at &#34;C&#34; was 5.1 inches, the radius r 3  at &#34;d&#34; was 5.1 inches and the radius r 5  at &#34;E&#34; was 5.5 inches. Consequently, the red blood cells accumulated at &#34;E&#34; instead of &#34;C&#34; where they were desired. By changing the radius r 4  to be 5.1 inches at &#34;C&#34;, r 5  to 4.9 inches at &#34;E&#34; and r 3  to 4.6 inches at &#34;D&#34;, the red blood cells accumulated at &#34;C&#34;. 
     Note that an increasing radius may be desirable for certain applications. For example, if it were desired to retain some plasma with packed red cells to lower the hematocrit, a pocket of plasma could be retained by first increasing the radius from the bottom of the bag (C and D) to midway up the bag and then decreasing the radius from the midpoint to the top of the bag (A and B). 
     We have also found that the position of the outlet port 45 at &#34;A&#34; with respect to the weight plate 15 and the back support member 9, is critical for complete removal of the desired component. The outlet port 45 must be positioned at an innermost radius as shown in FIG. 8A. If the port is allowed to fall back to the back support member due to the centrifugal force, some components may be trapped at an inner radius, as happened to component A in FIG. 8B. The outlet port may be held directly to the weight plate 15 with clips, or a deformable support member 100 may be used to position the outlet port 45 against the weight plate. This deformable support member 100 may be a spring mechanism or (as shown) may be shaped from a deformable material such as foam rubber. 
     A measure of the effectiveness of a wash procedure with respect to the removal of plasma proteins is the fraction of free hemoglobin removed as a function of the amount of wash solution used. The hemoglobin content of the fluid surrounding the RBC&#39;s is easily measured. Determining the hemoglobin content before and after a wash procedure provides a quantitative measure of the fraction of plasma proteins removed. 
     Our tests have shown that by repeating the wash cycle from 4 to 6 times, 97% of the plasma in the packed RBC&#39;s is removed (as measured by reduction of hemoglobin concentration). This procedure requires about 400 ml of wash solution and takes approximately 12 minutes. Comparatively, the IBM 2991 cell washing system removes 99.4% of the plasma [as measured by total protein) and consumes 1000 ml of saline (M. J. O&#39;Connor Wooten, Transfusion 16(5): 464-468 (1976)]and takes about 27 minutes (H. T. Meryman, et al., Transfusion 20(3): 285-292 (1980). 
     Equivalents 
     Those skilled in the art may recognize other equivalents to the specific embodiments described herein, which equivalents are intended to be encompassed by the claims attached hereto. 
     For example, instead of cell washing, the processing bag 8 may be used purely for pheresis (component separation) in which case, anticoagulated whole blood may be introduced at the lower corner &#34;C&#34; (port 47) and centrifugally separated into packed RBC&#39;s and plasma. After separation, the plasma would be expressed out corner &#34;A&#34; (port 45) in the manner previously described. 
     Also, the apparatus may be used for deglycerolization of frozen RBC&#39;s in glycerol. The frozen product is thawed and introduced into bag 8 at corner &#34;B&#34; (port 40) and processed as previously described until the glycerol is removed with the supernatant. 
     Furthermore, while the support member 9 and pressure plate 15 may be described in general as segments of a cylinder, they need not be cylindrically shaped but can be asymetric in shape to provide pooling of components at desired locations.