Abstract:
A method of collecting mononuclear cells, comprising separating whole blood into plasma and cellular components, combining the cellular components with plasma replacement fluid to form a first mixture, and separating the first mixture into mononuclear cells and at least one component.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Patent App. No. 62/277,198 filed Jan. 11, 2016, which is expressly incorporated herein by reference in its entirety. 
     
    
     FIELD OF THE DISCLOSURE 
       [0002]    The present disclosure is directed to fluid treatment systems and methods. More particularly, the present disclosure relates to systems and methods for separating blood into its constituents and subsequently treating and/or collecting the constituents. 
       BACKGROUND 
       [0003]    A variety of available blood processing systems allows for the collection and processing of particular blood components, rather than whole blood, from donors or patients. In the case of a blood donor, whole blood is drawn from the donor, a desired blood constituent separated and collected, and the remaining blood components returned to the donor. By removing only particular constituents rather than whole blood, it takes the donor&#39;s body a shorter time period to recover to normal blood levels, thereby increasing the frequency with which the donor may donate blood. It is beneficial to increase in this manner the overall supply of blood constituents made available for health care, such as red blood cells (RBCs), leukocytes, mononuclear cells (MNCs), plasma, and/or platelets, etc. In the case of a patient, whole blood is similarly drawn from the patient, a particular blood constituent first separated and then collected and/or treated, and the remaining blood components returned to the patient. The collected and/or treated blood constituent may be saved for future use, returned to the patient, and/or discarded and replaced with a suitable replacement. 
         [0004]    The separation of blood components from whole blood typically takes place prior to the collection or treatment of the separated blood component and may be achieved through a spinning membrane or centrifugation, in which whole blood is passed through a centrifuge or membrane after it is withdrawn from the patient. To avoid contamination and possible infection of the patient, the blood is preferably contained within a sealed, sterile fluid flow system during the entire separation process. Typical blood processing systems thus may include a permanent, reusable hardware assembly containing the hardware (drive system, pumps, valve actuators, programmable controller, and the like) that pumps the blood, and a disposable, sealed and sterile fluid circuit that is mounted in cooperation on the hardware. In the case of separation via centrifugation, the hardware assembly includes a centrifuge that may engage and spin a separation chamber of the disposable fluid circuit during a blood separation step. The blood, however, may make actual contact only with the fluid circuit, which assembly may be used only once and then discarded. In the case of separation via a spinning membrane, a disposable single-use spinning membrane may be used in cooperation with the hardware assembly and disposable fluid circuit. 
         [0005]    In the case of separation via centrifugation, as the whole blood is spun by the centrifuge, the heavier (greater specific gravity) components, such as red blood cells, move radially outwardly away from the center of rotation toward the outer or “high-G” wall of the separation chamber of the fluid circuit. The lighter (lower specific gravity) components, such as plasma, migrate toward the inner or “low-G” wall of the separation chamber. Various ones of these components can be selectively removed from the whole blood by forming appropriately located channeling seals and outlet ports in the separation chamber of the fluid circuit. 
         [0006]    In the case of separation via a spinning membrane, whole blood may be spun within a disposable spinning membrane, rather than within a separation chamber of a fluid circuit. Larger molecules, such as red blood cells, may be retained within one side of the membrane, while the smaller molecules, such as plasma, may escape through the pores of the membrane to the other side of the membrane. Various ones of these components can be selectively removed from the whole blood by forming appropriately located outlet ports in the housing of the membrane column. Various types of membranes with different pore sizes may be used, depending on the components to be separated. 
         [0007]    In the case of MNC collection, which includes the collection of lymphocytes, monocytes, and/or stem cells, MNCs can be removed from the whole blood of a patient, collected, and/or subjected to various therapies. Collected and treated MNCs may then be returned to the patient for the treatment of various blood diseases by, e.g., eliminating immunogenicity in cells, inactivating or killing selected cells, inactivating viruses or bacteria, reconstituting the immune system, and/or activating desirable immune responses. MNC treatments are used for blood or solid organ/tissue cancers, photopheresis treatments, autologous and allogeneic stem cell transplants, donor lymphocyte infusions, research collections, etc. 
       SUMMARY 
       [0008]    According to an exemplary embodiment, the present disclosure is directed to a method of collecting mononuclear cells, comprising separating whole blood into plasma and cellular components, combining the cellular components with plasma replacement fluid to form a first mixture, and separating the first mixture into mononuclear cells and at least one component. 
         [0009]    According to an exemplary embodiment, the present disclosure is directed to a method of collecting mononuclear cells, comprising separating whole blood into plasma and cellular components, purifying the plasma through a plasma adsorption column to create purified plasma, combining the cellular components with the purified plasma to form a first mixture, and separating the first mixture into mononuclear cells and at least one component. 
         [0010]    According to an exemplary embodiment, the present disclosure is directed to a method of collecting mononuclear cells, comprising providing an adsorption column through which whole blood is flowed to create purified whole blood, and separating the purified whole blood into mononuclear cells and at least one component. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]    Features, aspects, and advantages of the present embodiments will become apparent from the following description, appended claims, and the accompanying exemplary embodiments shown in the drawings, which are briefly described below. 
           [0012]      FIG. 1  is a diagrammatic depiction of a separation system useful in the separation and collection of mononuclear cells, according to an exemplary embodiment; 
           [0013]      FIG. 2  is a perspective view of the front panel of a separation system with a disposable processing set for collecting mononuclear cells mounted on the device, according to an exemplary embodiment; 
           [0014]      FIG. 3  is a diagram showing the disposable processing set of  FIG. 2 , according to an exemplary embodiment; 
           [0015]      FIG. 4  is a flow diagram illustrating an improved method for obtaining mononuclear cells, according to an exemplary embodiment; 
           [0016]      FIG. 5  is a flow diagram illustrating an improved method for obtaining mononuclear cells, according to another exemplary embodiment; and 
           [0017]      FIG. 6  is a flow diagram illustrating an improved method for obtaining mononuclear cells, according to another exemplary embodiment. 
       
    
    
     DETAILED DESCRIPTION 
       [0018]    There are several aspects of the present subject matter which may be embodied separately or together in the devices and systems described and claimed below. These aspects may be employed alone or in combination with other aspects of the subject matter described herein, and the description of these aspects together is not intended to preclude the use of these aspects separately or the claiming of such aspects separately or in different combinations as set forth in the claims appended hereto. 
         [0019]    Some embodiments may provide for collecting MNCs with reduced plasma lipid interference during MNC harvest. 
         [0020]    Some embodiments may provide for more accurate collection and harvest of MNCs by allowing for a clearer interface between blood component layers, 
         [0021]    During harvest of MNCs, non-target substances may be present in the MNC product that can interfere with accurate harvesting of the target MNCs. Plasma proteins and lipids may interfere, for example, in the event that the donor/patient has certain disease states or medications, such as elevated bilirubin levels and drugs such as mycophenolate mofetil (MMF) and cyclosporine, which cause hyperlipidemia. 
         [0022]      FIG. 1  is a diagrammatic depiction of a separation system  10  useful in the separation and collection of mononuclear cells, as described herein, and Fig,  2  shows an exemplary embodiment of the separation system  10 , The system  10  may include a separation component  12  and a disposable processing kit  14  ( FIG. 2 ) that is mounted thereon. Flow direction and rate may be controlled by a plurality of pumps  15  engaged with the processing kit  14 , In one embodiment, the separation principle used by the separator  12  is based on centrifugation, but an automated separator based on a different separation principle (e.g., spinning membrane, etc.) may also be used. 
         [0023]    A patient may be connected to the fluid circuit  14 , which may provide a sterile closed pathway between the separation component  12  and the remainder of the processing kit  14 , Whole blood that is withdrawn from the patient may be introduced into the separation component  12 , where the whole blood may be separated to provide a target cell population, which in the context of the present disclosure may be mononuclear cells. Other components separated from the whole blood, such as red blood cells and platelets may be returned to the patient or collected in pre-attached containers of the blood processing set. The separated target cell population, e.g., mononuclear cells, may then be collected for future use or prepared for various therapies. One example of a therapy involving MNCs that may benefit from reducing plasma lipid interference during MNC harvest is extracorporeal photopheresis or “ECP”. ECP involves the extracorporeal exposure of MNCs combined with a photoactive compound, such as 8-methoxypsoralen or “8-MOP” which is then photoactivated by ultraviolet light, followed by re-infusion of the treated MNCs. Removal of plasma lipids, which absorb UV light during irradiation, may lead to generally more consistent and less variable irradiation procedures, thereby enhancing accuracy of irradiation dosing and shortening procedure time. 
         [0024]    Apparatus useful in the collection of mononuclear cells, and providing the separation component  12  of  FIG. 1 , include for example the Amicus® Separator made and sold by Fenwal, Inc., of Lake Zurich, Ill. Mononuclear cell collections using a device such as the Amicus® are described in greater detail in U.S. Pat. No. 6,027,657, the contents of which are incorporated by reference herein in its entirety. The fluid circuit  14  ( FIG. 3 ) may include a blood processing container  16  defining a separation chamber suitable for harvesting MNCs from whole blood. 
         [0025]    As shown in  FIG. 2 , a disposable processing set or fluid circuit  14  (which includes container  16 ) may be mounted on the front panel of the separation component  12 . The processing set (fluid circuit  14 ) may include a plurality of processing fluid flow cassettes  23 L,  23 M and  23 R with tubing loops for association with peristaltic pumps  15  on the separation component  12 . Fluid circuit  14  may also include a network of tubing and pre-connected containers for establishing flow communication with the patient and for processing and collecting fluids and blood and blood components, as shown in greater detail in  FIG. 3 . 
         [0026]    As seen in  FIG. 3 , the disposable processing set  14  may include a container  60  for supplying anticoagulant, an in-process container  62 , a container  64  for holding a crystalloid solution, such as saline, a container  66  for collecting plasma, and a container  68  for collecting the mononuclear cells. 
         [0027]    With reference to  FIG. 3 , fluid circuit  14  may include inlet line  72 , an anticoagulant (AC) line  74  for delivering AC from container  60 , an RBC line  76  for conveying red blood cells from chamber  16  of set  14  to container  67 , a platelet-poor plasma (PPP) line  78  for conveying PPP to container  66  and line  80  for conveying mononuclear cells to and from separation chamber  16  and collection container  68 . 
         [0028]    The blood processing set may also include one or more venipuncture needle(s) for accessing the circulatory system of the patient. As shown in  FIG. 3 , fluid circuit  14  may include inlet needle  70  and return needle  82 . In an alternative embodiment, a single needle may serve as both the inlet and outlet needle. 
         [0029]    Fluid flow through fluid circuit  14  may be driven, controlled and adjusted by a microprocessor-based controller in cooperation with the valves, pumps, weight scales and sensors of separation component  12  and fluid circuit  14 , the details of which are described in the previously mentioned U.S. Pat. No. 6,027,657. 
         [0030]    A separation chamber may be defined by the walls of the processing container  16 . The processing container  16  may comprise two different compartments  16   a  and  16   b  ( FIG. 3 ). Using both compartments  16   a  and  16   b  for separation in a procedure may enable multiple target products to be separated simultaneously and/or multiple steps to be completed simultaneously. If only one compartment is used for separation, the other compartment may optionally be used as an in-process, waste, or storage container. In operation, the separation device  12  may rotate the processing container  16  about an axis, creating a centrifugal field within the processing container  16 . Details of the mechanism for rotating the processing container  16  are disclosed in U.S. Pat. No. 5,360,542 titled “Centrifuge with Separable Bowl and Spool Elements Providing Access to the Separation Chamber,” which is also incorporated herein by reference in its entirety. 
         [0031]    In one embodiment, an apheresis device or system  10  may include a programmable controller that is pre-programmed with one or more selectable protocols. A user/operator may select a particular processing protocol to achieve a desired outcome or objective. The pre-programmed selectable protocol(s) may be based on one or more fixed and/or adjustable parameters. During a particular processing procedure, the pre-programmed controller may operate the separator  12  and processing chamber  16  associated therewith to separate blood into its various components, as well as operate one or more pumps to move blood, blood components and/or solutions through the various openable valves and tubing segments of a processing set, such as processing set  14  illustrated in  FIG. 3 . The various processing steps performed by the pre-programmed automated apheresis device may occur separately, in series, simultaneously or any combination of these. 
         [0032]    An automated apheresis device may be used to perform MNC collection in a batch process in which MNCs continuously collect in the chamber  16  until the target cycle volume is reached. During the continuous collection of MNCs within the chamber  16 , different blood components separate into layers that may be detected by an optical interface detector that monitors the location and presence of the interface between layers. Details of an exemplary mechanism for interface detection are disclosed in U.S. Pat. No. 6,027,657, the contents of which are incorporated by reference herein in its entirety. Before and during the transfer of the MNCs out of the chamber  16 , MNCs and other blood components (e.g., plasma, etc.) may pass through an optical sensor  17 , located downstream of the chamber  16 , which detects the presence of cells in the tubing line to determine the start and end of the MNC harvest (i.e. when to open and close the valves leading to the product container). The term “downstream” describes an event proximal to post-separation, and the term “upstream” describes an event proximal to pre-separation. “Downstream” and “upstream” are relative terms, with the reference point being the time/location of separation. After MNC harvest is complete, the remaining cells in the line may be flushed into the product container with a predetermined volume of plasma known as the “plasma flush”. 
         [0033]    The ability of the interface detector to accurately detect the interface between blood component layers may be facilitated by removal of non-target substances (e.g., plasma proteins and lipids) that may be present in the blood that can interfere with the separation procedure. Additionally, the removal of non-target substances may improve the ability of the optical sensor  17  to accurately detect the presence of cells in the tubing line to determine the start and end of the MNC harvest to facilitate precise harvesting of the target MNCs. 
       EXAMPLES 
       [0034]    Without limiting any of the foregoing, the subject matter described herein may be found in one or more methods, systems and/or products. For example, in a first aspect of the present subject matter, an improved system and method for obtaining MNCs is set forth in  FIG. 4 . The inlet needle  70  of  FIG. 3  attached to inlet line  72  may first be connected to a blood source  5  (e.g, donor, patient, blood bag, etc.). Whole blood may enter the separation chamber  16  of the separator  12 , which separates the whole blood into plasma and cellular components. The plasma may be separated and directed into a plasma container  66 , and the cellular components may be separated and directed into a different container  67 . The separated cellular contents may be combined with a replacement fluid  69  that has minimal non-target content (e.g., plasma proteins and/or lipids) that may interfere with optical sensor readings. Examples of suitable replacement fluids include fresh frozen plasma, immunoglobulin solution, albumin, and/or other colloid solutions. The cellular components mixed with replacement fluid may then be returned to the separation chamber  16 , where target MNCs may be collected. The target MNCs may be harvested into a designated container  68  to be processed for further treatment. Non-target components may be collected or returned to the patient/donor, 
         [0035]    The process and steps of whole blood initially entering the separation chamber  16  and the cellular components and replacement fluid mix returning to the separation chamber  16  portrayed in  FIG. 4  may take place substantially in series if only one compartment  16   a  or  16   b  is utilized. Alternatively, the process and steps of whole blood entering the separation chamber  16  and the fluid mix returning to the separation chamber  16  may take place substantially at the same time if both compartments  16   a  and  16   b  are utilized. In an embodiment in which the processes take place substantially in series, whole blood entering one of the compartments  16   a  or  16   b  may separate into plasma and cellular components, both of which may be directed to separate containers until separation of plasma and cellular components is complete. An optical sensor  17  may optionally be placed downstream of the separation chamber  16   a  and/or  16   b  at a tubing line leading to the MNC product container  68  and/or leading to the plasma container  66  to determine when plasma is clear enough and plasma diversion can stop. Subsequently, the cellular components may combine with the replacement fluid within one of the compartments  16   a  or  16   b,  and further separation into target MNCs and non-target components may take place, 
         [0036]    In an embodiment in which the steps of whole blood entering the separation chamber  16  and the fluid mix returning to the separation chamber  16  take place substantially at the same time, whole blood entering a first compartment (e.g.,  16   a ) may separate into plasma and cellular components, with the plasma being sent to an plasma container  62 . Simultaneously, the cellular components may join the replacement fluid and together enter a second compartment (e.g.,  16   b ) and there further separate into target MNCs and non-target components. An optical sensor  17  may optionally be placed downstream of the separation chamber  16   a  at a tubing line leading to the MNC product container  68  and/or leading to the plasma container  66  to determine when plasma is clear enough and plasma diversion can stop. As the replacement fluid continues to enter compartment  16   b  and the clarity of the plasma leaving compartment  16   a  improves sufficiently as determined by the optical sensor  17 , the plasma diversion from compartment  16   a  into the plasma container  66  can be stopped, and any unseparated whole blood, including the contents of compartment  16   a,  may be directed to compartment  16   b  to continue MNC collection. 
         [0037]    In another aspect of the present subject matter, an improved method for obtaining MNCs is set forth in  FIG. 5 . An inlet needle may first be connected to a blood source  5  (e.g, donor, patient, blood bag, etc.) at step  100  of  FIG. 5 . In step  200 , whole blood enters a separator, which separates the whole blood into plasma (step  300   a ) and cellular components (step  300   b ). In the embodiment in  FIG. 5 , the separator of step  200  may be a centrifugal or spinning membrane separator. An exemplary spinning membrane and hardware is disclosed in greater detail in PCT Patent Application No. PCT/US2012/28492, which is incorporated herein by reference in its entirety, although any suitable membrane assembly may be used, Plasma separated in step  300   a  may be flowed through an adsorption column in step  400 . An exemplary adsorption column is the MONET filter made and sold by Fresenius Medical Care. Another exemplary adsorption column is disclosed in greater detail in International Publication No, WO 2012/141697 and U.S. Pat. No, 6,569,112, each of which is hereby incorporated by reference herein in its entirety, although any suitable column may be used. In step  500 , purified plasma from the adsorption column in step  400  may be combined with the cellular components of step  300   b.  The combined product of step  500  may then be separated in a separation chamber of a separator in step  600  to collect and harvest MNC target cells separated from non-target components. 
         [0038]    In another aspect of the present subject matter, an improved method for obtaining MNCs is set forth in  FIG. 6 . An inlet needle may first be connected to a blood source  5  (e.g, donor, patient, blood bag, etc.) at step  101  of  FIG. 6 . In step  201 , whole blood enters a whole blood adsorption column, which removes certain lipids, proteins, antibodies, and/or fatty acids in step  301 . An exemplary whole blood adsorption column is the DALI® adsorber made and sold by Fresenius Medical Care, although any suitable whole blood adsorption column may be used. Purified whole blood exiting from the WB adsorption column of step  201  may then be separated in a separation chamber  16  of a separator in step  401  to collect and harvest MNC target cells separated from non-target components. 
         [0039]    The embodiments disclosed herein are for the purpose of providing a description of the present subject matter, and it is understood that the subject matter may be embodied in various other forms and combinations not shown in detail. Therefore, specific embodiments and features disclosed herein are not to be interpreted as limiting the subject matter as defined in the accompanying claims.