Patent Publication Number: US-11660603-B2

Title: Cell concentration devices and methods including a syringe and a syringe holder

Description:
RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 16/835,053, filed on Mar. 30, 2020, which is a divisional of U.S. application Ser. No. 14/764,115, filed Jul. 28, 2015, now U.S. Pat. No. 10,603,665, which is the U.S. National Stage of International Application No. PCT/US2014/013636, filed on Jan. 29, 2014, published in English, which claims the benefit of U.S. Provisional Application No. 61/757,993, filed on Jan. 29, 2013 and U.S. Provisional Application No. 61/897,587, filed on Oct. 30, 2013. The entire teachings of the above applications are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     In the field of regenerative medicine, access to a broad cross section of sub-dermal tissue is typically required to not only source cells but to also deliver therapy. Fluid tissue that is aspirated or otherwise sourced is often separated into one or more components that are present in the fluid tissue, e.g., plasma, red blood cells, fat cells, stem cells or other nucleated cells. Typically, one or more selected components of the fluid tissue are concentrated into a small volume so that the selected components can be used clinically. For example, there are several commercial devices to separate and concentrate nucleated cells from aspirated bone marrow, fat, or cord blood. Some of these systems employ a floating insert or buoy that is meant to create an interface between the separated fluid components or fractions of interest. The challenge for any apparatus designed to accomplish such a task is the ability to volume reduce the fluid in which the nucleated cells are suspended while recovering as many cells as possible. For example, in marrow aspirate, approximately 1 to 2 percent of the cells suspended in the fluid are the target nucleated cells. Many commercial devices are not able to consistently capture high percentages of nucleated cells while at the same time efficiently volume reduce (i.e., concentrate) the beginning fluid. In other words, many devices are not able to simultaneously obtain a high yield and a high final concentration. 
     Apparatus and methods for separating components of different densities from a physiological fluid containing cells are described in a previously filed application, International Application No. PCT/US2010/036696, filed on May 28, 2010, published on Dec. 2, 2010 as WO 2010/138895 A2, and incorporated herein by reference in its entirety. 
       FIG.  1    is a diagram illustrating a separation system  1700  showing different components of a fluid inside the container  1702  after centrifugation. After centrifugation, the least dense fluid  2000  will be above the insert  1300 . The insert can be made of a material of a certain density such that after centrifugation of blood, including blood from marrow, the insert spans the space between the least dense plasma  2000  and the dense red cells  2004 , with the intermediate dense material  2002 , e.g., nucleated cells, residing in the upper funnel-shaped portion  1304  of the insert. The separation system  1700  can include a vent  1716  disposed in the top  1706  of the container  1702  and a fluid port  1718  disposed in or adjacent the top  1706 . The air vent  1716  can prevent a vacuum from being created when fluid is withdrawn from the container  1702 . A cannula assembly  1500  with a closed end  1502  is inserted through the injection port  1714 . Before insertion of the cannula assembly, a clamp  1800  can be applied to sidewall of container  1702  to hold the insert  1300  in place during subsequent fluid extraction. The closed end  1502  of the cannula assembly butts against the insert and closes the through hole  1308  of the insert. The closed end of the cannula assembly  1500  and the insert can form a seal, thus isolating denser fluid component or components beneath the seal from fluid components above the seal. The cannula assembly includes two cannulae, an inner cannula  1508  and an outer cannula  1510 , that fit coaxially into each other. 
     As shown in  FIG.  1   , the cannula assembly includes a series of two parallel side holes or ports in the two cannulae to line up at different predetermined heights above the closed distal end  1502 . A first set of side ports  1506  can be located near the closed distal end  1502 . A second set of side ports  1504  can be located above the upper funnel-shaped portion  1304  of insert  1300 . Fluid above the distal end  1502  of the cannula assembly can be removed in at least two fractions or components based on these two different predetermined heights. Fluid can be removed through the cannula assembly  1500  into connected syringes  1802 ,  1804  using valve  1806 . For example, when the top side ports  1504  are aligned and opened, fluid above the top side ports can be extracted into a first syringe  1802  through inner cannula  1508 . By rotating the two cannulae with respect to each other, the top side ports in the cannula assembly  1500  are misaligned and sealed off, while the bottom side ports are aligned and opened. As shown in  FIG.  1   , the side ports may be radially offset by 90 degrees, requiring a relative rotation of 90 degrees to change which ports are aligned. When the bottom side ports  1506  are located just above the seal created by the closed end  1502  of the cannula assembly, substantially all fluid above the seal, but below the top side ports, can be extracted through inner cannula  1508  into a second syringe  1804 . 
       FIGS.  2 A- 2 C  are a series of sequential diagrams illustrating the extraction of fluid components using a separation system  2100 , the system including a container  2102  having a movable bottom or plunger  2104 . Centrifugation separates the fluid in the container by density into separate components or fractions.  FIG.  2 A  illustrates the position of an insert, such as insert  2600 , in relation to three components of a fluid in the separation system  2100  after centrifugation. The components are a low density fraction  2000 , such as plasma, a medium density fraction  2002 , such as buffy coat or nucleated cells, and a high density fraction  2004 , such as red blood cells. 
     To retrieve the separated layers or fluid components, the user takes the syringe or container  2102  out of the centrifuge. As shown in  FIGS.  2 A- 2 B , the user then uncaps the luer connector of port  2118 , attaches a plasma extraction syringe  2300 , and pulls back on the plunger. The calculated combination of 1) the fluid flow of plasma as it is being evacuated from the collection syringe or container  2102 , which can be lateral to the center injection port, 2) the size of the center hole  2608  or holes  2610  in the insert  2600 , 3) the relative density of the different fluids inside the container  2102 , and 4) the forces required to extract fluid of different densities under these known parameters, results in substantially only plasma moving into the plasma syringe  2300 . Because the air vent  2116  is capped, the collection syringe or container  2102  is a vacuum. Thus the movable bottom or plunger  2104  and the insert  2600  rise in the collection syringe or container  2102  as the plasma is extracted. The target cells, such as buffy coat, stay in the through hole or holes  2610  of the insert  2600 . 
     As shown in  FIGS.  2 B- 2 C , after removal of the plasma  2000 , the insert  2600  has risen to the top of the syringe or container  2102  and effectively seals off port  2118  connected to the plasma extraction syringe. At this point the user uncaps the air vent  2116  making the collection syringe or container  2102  no longer under vacuum pressure. A second target cell extraction syringe  2302  with a cannula  2400  attached is then inserted through the center injection port  2114 . Since 1) the insert  2600  always ends up at the top of the collection syringe or container  2102  after removal of the plasma and 2) the height of the insert  2600  is known, then the distance between the top of the injection port  2114  and bottom of the through holes  2610 ,  2608  of the insert  2600  is always the same after removal of the plasma. The length of the cannula  2400  is such that it reaches just to the bottom of the center through hole  2608  in the insert  2600  after removal of the plasma. Thus, when the user pulls back on the plunger of the target cell extraction syringe  2302 , after the air vent  2116  has been uncapped, the target cells residing in the through hole or holes  2610 ,  2608  are removed. 
     SUMMARY OF THE INVENTION 
     A system for separating components of different densities from a physiological fluid containing cells using a centrifuge includes a container having a top, a sidewall extending from the top, and a bottom disposed opposite the top and in sealing engagement with the sidewall. The container defines a cavity for receiving the fluid. The system includes an insert slidably disposed in the cavity of the container. The insert defines a lumen through the insert, the lumen including a hole and a funnel-shaped upper portion in fluid communication with the hole. The lumen forms an open fluid path between opposite ends of the insert. The insert has a density such that upon centrifugation a selected component of the fluid resides within the lumen. A container port is disposed in the top of the container to transfer the fluid into the container and to withdraw a fluid component other than the selected component from the container. The system further includes a manifold that includes a manifold port, a vent to vent the container, and a connector to couple to the container port. A cannula is receivable in the manifold port and extendable through the container port into the container and into the lumen of the insert to withdraw the selected component from the lumen. 
     The cannula can include a closed end to close the hole in the insert and a side port to withdraw the selected component. The cannula and the insert may form a seal when the closed end of the cannula closes off the hole in the insert. In an embodiment, the cannula a first cannula, and the system further includes a second cannula extendable through the container port to withdraw the component other than the selected component. The second cannula may be receivable in the manifold port. The system may include two manifolds, each including a manifold port, a vent to vent the container, and a connector to couple to the container port, and the first cannula can be receivable in the manifold port of one manifold while the second cannula can be receivable in the manifold port of the other manifold. 
     The container can be a syringe and the bottom can be movable, i.e., a plunger, which can have a removable handle. In an embodiment, the plunger is a first plunger and the system further includes a second plunger disposed in the syringe below the first plunger to move the first plunger, for example, to transfer the fluid into the container. The system may further include a clamping mechanism to hold the insert in place after centrifugation, the clamping mechanism being configured to press the sidewall of the container inward against the insert. 
     A method of separating components of different densities from a fluid containing cells using a centrifuge includes receiving the fluid in a separation system such as the separation system described above, and applying centrifugal force to the separation system. The method further includes, after centrifugation, withdrawing a fluid component other than the selected component through the container port; coupling a manifold to the container port, the manifold including a manifold port and a vent to vent the container; extending a cannula through the container port into the container and into the lumen of the insert, the cannula receivable in the manifold port; and withdrawing the selected component with the cannula from the lumen of the insert. 
     Withdrawing the selected component may include withdrawing the selected component through a side port in the cannula. In an embodiment, the cannula is a first cannula and withdrawing the component other than the selected component includes extending a second cannula through the container port, the second cannula receivable in the manifold port, and withdrawing the component other than the selected component with the second cannula. The manifold can be coupled to the container before the withdrawing of the component other than selected component. The method may further include with a clamping mechanism, holding the insert in place after centrifugation. 
     A system for separating components of different densities from a physiological fluid containing cells using a centrifuge includes a container, having a bottom, a top disposed opposite the bottom, and a sidewall extending from the top, the container defining a cavity for receiving the fluid. An insert is slidably disposed in the cavity and defines a lumen through the insert, the lumen including a hole and a funnel-shaped upper portion in fluid communication with the hole. The insert has a density such that upon centrifugation a selected component of the fluid resides within the lumen. The lumen forms an open fluid path between opposite ends of the insert. A container port is disposed in the top of the container. An extraction cap is provided to couple to the top of the container, the extraction cap including a cannula assembly receivable in the container port. The cannula assembly is extendable into the cavity of the container to butt against the insert and to withdraw the selected component from the lumen of the insert. 
     The cannula assembly can include an inner cannula coaxially disposed within an outer cannula. The inner cannula may include a closed end to close the hole in the insert and a side port to withdraw the selected component, the inner cannula and the insert forming a seal when the closed end of the inner cannula closes off the hole in the insert. The outer cannula may include an open end displaced from the distal end of the cannula assembly to withdraw fluid at a predetermined height above the distal end of the cannula assembly. 
     In an embodiment, the extraction cap includes a first port in fluid communication with the inner cannula and a second port in fluid communication with the outer cannula. The system may further include a first syringe to couple to the first port and a second syringe to couple to the second port. The cap may include an assembly tab adjacent the first and second ports, the assembly tab extending from the cap to prevent the second syringe from coupling to the first port. The system may further include a lock-out element on the second syringe. For example, the lock-out element includes a tab that locks a plunger of the first syringe until second syringe is removed from the cap. In an embodiment, the extraction cap includes an outer part and an inner part, the inner part carrying the needle assembly and being movable relative to the outer part. The cap may include a locking screw coupled to the outer part and positioned at an angle relative to inner part to push the inner part toward the container with rotation of the locking screw. 
     A method of separating components of different densities from a fluid containing cells using a centrifuge includes receiving the fluid in a separation system, the system including a container having a bottom, a top disposed opposite the bottom, and a sidewall extending from the top, the container defining a cavity for receiving the fluid. A container port is disposed in the top of the container. An insert is slidably disposed in the cavity of the container, the insert including a funnel-shaped upper portion and a hole therethrough, the insert having a density such that upon centrifugation a selected component of the fluid resides within the upper portion of the insert. The method includes applying centrifugal force to the system and inserting a cannula into the container through the container port to butt against the insert, the cannula having one or more side ports displaced from a distal end of the cannula. The method further includes withdrawing the selected component through the side ports in the cannula; ejecting at least a portion of the withdrawn component through the side ports causing one or more fluid jets in the funnel-shape upper portion of the insert to release cells that adhere to the insert; and withdrawing the ejected portion and cells released by the fluid jets through the side ports. 
     In any of the systems or methods described herein the insert can be rigid. The volume contained in the lumen upper portion of the insert can be between 5% and 20% of the volume of the container cavity. The selected component (also referred to herein as a target fraction) can be buffy coat and the component other than the selected component can be blood plasma. 
     Embodiments of the current invention overcome the limitations of known devices for concentration of cells sourced from marrow or other tissue. For example, the insert of the separation device does not form a closed recess or a depression or indent to capture cells, but rather allows for the natural sedimentation of the fluid within the container and through the insert. The insert defines a lumen that has at least one relatively large through hole or channel, including a funnel-shaped upper portion, that allows for the free flow of fluid within the container and through the insert and does not interfere with the natural layering of different density components of the fluid. In addition, the insert identifies the location of a layer of interest, including the target cells. The funnel-shaped upper portion and the through hole reduce the cross-sectional area and increase the thickness of the layer of interest. This facilitates extraction of the target cells and contributes to a high yield and high concentration of the target cells. 
     Embodiments of the current invention overcome limitations of other systems that use inserts or buoys without a through hole and where the fluid path under centrifugation is confined to the distance between the inner wall of a container or tube and the outer walls of the inserts or buoys. In those systems, minor clots, particles, or other inconsistencies in the fluid can lodge between the walls of the tube and the buoys interfering with the natural layering of the different density components of the fluid. The insert(s) described includes a density selected such that after centrifugation the target cells reside within the hole, the funnel-shape upper portion, or lumen defined by the insert. Under gravitational force the insert floats freely within the container with substantially all of the fluid flowing through the hole or lumen of the insert, but not between the outer wall of the insert and the inner wall of the container. The distance between the inner wall of the container and the outer wall of the insert creates enough space to allow the insert to move freely within the container. 
     Embodiments of apparatus and methods for separating components of a fluid can be combined with devices and methods to access and source, e.g., aspirate, tissue, such as the aspiration needle assemblies described in International Application No. PCT/US2010/036696. Once tissue is sourced, e.g., loaded into a separation system, the system can be centrifuged. Upon centrifugation, the target cells naturally sediment into the through hole or lumen of the floating insert. These cells are then isolated by means of a cannula. The closed end of the cannula can close the hole in the insert. The target cells residing in the hole of lumen of the floating insert may be sealed from fluid below while fluid above the insert is removed through a cannula. The combination of the cell concentration and separation apparatus described herein with an aspiration apparatus allows a clinician the ability to access subcutaneous tissue in a less traumatic manner and then concentrate nucleated cells from that tissue aspirate. The apparatus can be combined, e.g., coupled or connected, by means of tubing and fluid ports, including luer connections, to create a total solution from aspiration to concentration. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention. 
         FIG.  1    illustrates a separation system showing different components of a fluid after centrifugation. 
         FIGS.  2 A- 2 C  are a series of sequential diagrams illustrating the extraction of fluid components using a separation system having a movable bottom. 
         FIG.  3    is an exploded view of a system for separating components of difference densities from a physiological fluid according to an example embodiment of the invention. 
         FIG.  4    is a perspective view of a container including an insert according to an example embodiment of the invention. 
         FIG.  5    is a perspective view of the container of  FIG.  3    positioned in a base including a clamping mechanism. 
         FIG.  6    is a side view of an extraction cap according to an example embodiment of the invention. 
         FIG.  7    is a side view of a system including the container and base of  FIG.  5    and the extraction cap of  FIG.  5   . 
         FIG.  8    is a sectional view of the system of  FIG.  7   . 
         FIG.  9    illustrates a needle assembly positioned in an insert. 
         FIG.  10 A  illustrates a system for concentrating cells according to another example embodiment of the invention. 
         FIG.  10 B  is a perspective view of a double plunger syringe including an insert. 
         FIGS.  11 A- 11 C  illustrate movement of the plungers of the double plunger syringe of  FIG.  10 A . 
         FIG.  12    illustrates inserts for use with a system for concentrating cells according to example embodiments of the invention. 
         FIG.  13    illustrates the collection syringe of  FIG.  11    positioned in a holder including a clamping mechanism. 
         FIG.  14 A  illustrates elements of the system of  FIG.  10 A  arranged for extraction of fluid components from the container. 
         FIG.  14 B  is a detailed view of the cannula and manifold of  FIG.  14 A . 
         FIG.  15    illustrates a cannula positioned against an insert, the cannula including a side port displaced from the distal end of the cannula to withdraw fluid at a predetermined height above the distal end, e.g., above the insert, to withdraw substantially only plasma. 
         FIG.  16    illustrates a cannula positioned against an insert, the cannula including a side port displaced from the distal end of the cannula to withdraw fluid at a predetermined height above the distal end, e.g., within the insert, to withdraw substantially all of the target fraction. 
         FIG.  17    illustrates an alternative embodiment including an extraction syringe coupled to a collection syringe for withdrawing a fluid component other than a selected component, e.g. for withdrawing plasma. 
         FIG.  18    illustrates a separation system according to another example embodiment of the invention. 
         FIG.  19    illustrates the separation system of  FIG.  18    showing different components of a fluid after centrifugation. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A description of example embodiments of the invention follows. 
     A prior version of a system for concentrating and separating cells (also referred to herein as a cell concentrator) has been described in International Application No. PCT/US2010/036696, published on Dec. 2, 2010 as WO2010/138895, incorporated herein by reference in its entirety. Here, an improved system is described that has been fabricated and tested. Some key features of the improved system include: 
     a) a locking base; 
     b) a double needle extraction system with jet flushing; 
     c) an extraction needle lock that forces needle to seat in float; and 
     d) a syringe lock-out tab to ensure proper order of extraction. 
       FIG.  3    is an exploded view of an improved system  10  for separating components of difference densities from a physiological fluid according to an example embodiment of the invention. System  10  includes a base  12 , a separation vial (container)  14 , a float (insert)  16  disposed in the vial  14 , and an extraction cap  18  with syringes  20 ,  22  and a cannula assembly including extraction needles (cannulae)  24 ,  25 . 
     To separate buffy coat from red blood cells and plasma, the float  16  should have a density that matches that of the buffy coat, whose density is roughly 1.06 g/cc. A polystyrene material was selected to manufacture the insert because the material&#39;s density is close to that of buffy coat. However, testing indicated that a polystyrene float was lighter than desired. Small slugs  56  ( FIG.  9   ), e.g., pieces of stainless steel, can be added into the float to adjust its density. The process of testing a device in blood to ensure that the density matches the desired density can be repeated for new batches of plastic, e.g., during manufacturing, or for testing different materials. 
     The materials of the system are polycarbonate for most of the parts with the vial being PET (Polyethylene teraphthalate plastic) and the float polystyrene, as described above. Other suitable materials for the float may be polyethylene or polypropylene materials which tend to be less sticky to cells than polystyrenes. However, the densities of these materials are lower than that of buffy coat, so a larger metal material may need to be incorporated into the float to achieve the desired density. Another option is to coat the surface of the float and the inside of the vial with a substance that prevents cells form sticking, for example, a coating from the company Hydromer. 
       FIG.  4    is a perspective view of container  14  including an insert  16  according to an example embodiment of the invention. Container  14  is a vial that has a bottom  13 , a top  17  disposed opposite the bottom, and a sidewall extending from the top, the top including a lid  19  to close the container. The vial defines a cavity for receiving fluid. A physiological fluid containing cells, such as blood or marrow, is introduced into the separation vial  14  via a port  28  prior to centrifugation. After the fluid is centrifuged, the separation vial  14  is placed into the base  12 . As shown in  FIG.  5   , the base  12  has a lever  26  with a cam  27  that locks the vial to the base by deforming the vial wall  15 . This deformation locks the float  16  inside the vial  14  in place so that it cannot move. In other words, the cam and lever of base  12  operate as a clamping mechanism that, when engaged, prevents movement of the float. Movement of the float, such as during the extraction procedures described below, can disrupt the target cell layer, e.g., the buffy coat. 
     Post centrifugation, the serum (plasma) is above the float  16 , the red blood cells are below the float and the target cells (e.g., buffy coat) are inside the float funnel. This separation of fluid components is analogous to what is shown in  FIG.  1   . The cap or lid  19  of the vial  14  includes a silicone septum  30  and air vents  32 . As shown in  FIGS.  4  and  5   , four air vents  32  are arranged around the septum  30  and four protrusions  60  extend from the lid  19 . The extraction needles (cannulae)  24 ,  25  are inserted through the septum  30 , which functions as a port into the container. The air vents are included so that one does not pull a vacuum when extracting the target cells or the plasma serum. 
       FIG.  6    is a side view of an extraction cap  18  according to an example embodiment of the invention. Extraction cap  18  is distinct from the lid  19  configured to couple to the top of the vial (container)  14 . The extraction cap includes the cannula assembly, including needles (cannulae)  24  and  25 , which are receivable in container port  30 . 
     As illustrated in  FIGS.  7  and  8   , the extraction cap  18  includes an outer part  31  and an inner part  33  movable with respect to the outer part. The outer part  31 , which includes apertures  62  to engage the protrusions  60  of the lid, snaps onto the top  17  of the vial  14 . The inner part  33  includes two ports  36 ,  38  for connecting the two syringes  20 ,  22  to the inner and outer needles (cannulas) of the extraction cannula assembly. An assembly tab  42  on the inner part  33  ensures that the larger syringe  20  can only be connected to the port that is in fluid communication with the outer extraction needle  25 . 
     As shown in  FIG.  7   , a lock-out tab  44  is mounted on the large syringe  20  that forces the user to activate the large syringe first. The lock-out tab  44  blocks the plunger of syringe  22 . Once syringe  20  is filled with serum extracted from vial  14 , syringe  20 , including lock-out tab  44 , is removed and the small syringe  22  may then be activated. This ensures the proper order is followed when extracting fluid components from vial  14 . 
     To remove the target cells, the extraction cap  18  is placed over the vial  14 , as illustrated in  FIGS.  7  and  8   . The extraction needles  24 ,  25  are advanced through the silicone septum (container port)  30  and into the vial  14 . The extraction cap  18  snaps onto the lid of the vial. Once in place, the syringe/needle assembly is pushed down by the operator so that the distal end of the needle assembly bottoms out into the funnel in the float  16  ( FIG.  9   ). The locking screw  34  is then tightened. This screw is at an angle. As the screw is tightened, it slightly pushes the needle assembly down to ensure that it fully engages the bottom of the float. In the embodiment shown in  FIGS.  7  and  8   , the locking screw  34  is positioned at a small angle within the horizontal plane. In the embodiment of  FIG.  3   , the locking screw  34  is angled out of the horizontal plane to perform the same function. 
     As shown in  FIGS.  8  and  9   , the extraction cannula assembly includes an inner needle (inner cannula)  24  and an outer needle (outer cannula)  25 . The open distal end  46  of outer needle  25  is positioned at a set distance from the distal end  48  of the inner needle  24  such that the outer needle sits at or near the top of the float  16 . The outer needle is used to extract the serum (plasma).  FIG.  9    illustrates the needle assembly positioned in the float (insert)  16 . As shown, the inner needle  24  has a distal taper and multiple holes (ports)  40 , e.g.,  3  side holes, at or near its distal end  48 . The inner needle  24  sits in the bottom of the float and plugs hole  52  in the float. The extraction process starts with the outer needle  25 , which is in fluid communication with large syringe  20 . The serum is removed until bubbles are seen in the syringe  20 . Then, the selected component (e.g., stem cells, buffy coat) is removed using the small syringe  22  and the inner needle  24 , the selected component being withdrawn through holes  40  of inner cannula  24 . As cells of the selected component adhere to the inner wall  54  of the float (insert)  16 , the cell solution that has been removed, or portion thereof, is pushed back, e.g., ejected, into the float through the holes  40  causing one or more jets of fluid in the funnel-shaped portion of the float. This jet flushing releases the cells from the float wall  54 . The cells are then sucked up during a second withdrawal into the syringe  22 . 
     Embodiments shown in  FIGS.  3 - 9    and described herein have many advantages. One advantage of the double cannula extraction needle is that the operator only has to push the cannula assembly through the silicone septum of the separation vial once, which reduces the risk of mixing the separated fluid components in the vial. Other systems require needles or cannulae to be inserted serially, which carries a higher risk of infection and of moving the target cells to be extracted. Another advantage is the extraction needle lock, which includes a locking screw that, when tightened, drives the cannula assembly down into the float to ensure that the extraction needle (inner cannula) fully engages the bottom of the float. The locking screw of the needle lock may be positioned at an angle with respect to the extraction needle, such that rotation of the screw drives the extraction needle down. The screw may be at an angle out of the horizontal plane ( FIG.  3   ), or at an angle within the horizontal plane, as illustrated in  FIGS.  6 - 8   . Furthermore, the lock-out tab on the larger syringe prevents an operator from using the small syringe first, thereby ensuring proper order of withdrawal of fluid components. 
     A system for separating components of different densities from a fluid containing cells and for concentrating cells according to another example embodiment of the invention is described below and illustrated in  FIGS.  10 A- 17   . A system for concentrating bone marrow or blood is described, although the principles of the invention can be applied to other fluids, including other physiological fluids. The system uses a container having a movable bottom, e.g., a syringe with at least one plunger, for both the collection and centrifugation of the fluid specimen. 
     As illustrated in  FIGS.  10 A and  10 B , a system  100  for separating components of different densities from a fluid containing cells using a centrifuge includes a collection syringe (container)  114  having a top  117 , a sidewall  115  extending from the top, and a bottom  113 , e.g., a plunger, disposed opposite the top and in sealing engagement with the sidewall. The container defines a cavity for receiving the fluid. An insert  116  is slidably disposed in the cavity of the container  114 . Similar to insert  16  described above in reference to  FIG.  9   , insert  116  defines a lumen through the insert, the lumen including a hole  52  and a funnel-shaped upper portion  50  in fluid communication with the hole. The lumen forms an open fluid path between opposite ends of the insert. The insert has a density such that upon centrifugation a selected component of the fluid resides within the lumen. A container port  130 , e.g., a luer connector, is disposed in the top of the container  114  to transfer the fluid into the container and to withdraw a fluid component other than the selected component from the container. As illustrated in  FIG.  10 A , the system further includes at least one manifold  160  that includes a manifold port  162 , a vent  164  to vent the container, and a connector  166  to couple to the container port  130 . A cannula  170 ,  172  ( FIG.  14 A ) is receivable in the manifold port  162  and extendable through the container port  130  into the container  114  and into the lumen of the insert  116  to withdraw the selected component from the lumen or a component other than the selected component. 
     As illustrated in  FIG.  10 A , the system can include a clamping mechanism  112  that can also double as a syringe holder. The container of the system can be a syringe that includes a plunger having a removable handle (not shown) or a syringe  114  with two plungers as shown, plunger  123  having a handle and plunger  113  being without a handle. A 30 ml plasma extraction syringe  120  is connected to a cannula  170  that fits through upper injection port  162 , manifold  160  and lower luer connection  166 . A 5 ml concentrate extraction syringe  122  is connected to a cannula  172  that fits through upper injection port  162 , manifold  160  and lower luer connection  166 . The system can include a syringe holder  174  with washer on bottom and, optionally, O-ring  176 . For convenience of the user, the system can include two manifolds  160 , as illustrated in  FIG.  10 A . One manifold  160  receives cannula  170 ; the other manifold receives cannula  172 . A standard 4-way manifold may be used for each manifold  160 . The standard manifold includes 3 fluid channels and a switch (e.g, valve)  168 . Using switch  168 , each of the fluid channels can be selective closed or all channels can be open. Here, all channels are open. Vent  164  Coupled to the manifold can be a vent that has a micron filter. This way, a sterile environment can be maintained while venting during the extraction of fluid. The injection port  162  and the connector  166  can be swabable luer ports, which can be swabbed or wiped, e.g., with alcohol, for good sterile procedure. The swabable luer ports typically include an elastomeric (e.g., rubber) membrane that includes a slit that is normally closed, but parts when a cannula or male luer connector is inserted. Such ports are beneficial when connections are to be air tight. 
     A step by step overview of the operation of the system will be described. The first step is to use the double plunger syringe  114  (alternatively, a syringe with a removable handle) to fill the syringe with the blood or marrow specimen to be concentrated.  FIGS.  11 A- 11 C  illustrate movement of the plungers of the double plunger syringe  114 , such as during filling of the syringe with physiological fluid. It should be noted that during filling, the syringe port  130  is coupled to another container containing the source tissue or to a tissue aspiration needle (not shown). Pulling back on the second plunger  123  via its handle forces the first plunger  113  under vacuum pressure to move back and the syringe is thus filled. The second plunger  123  can be completely removed from the barrel of syringe  114  leaving behind the first plunger  113  that is not connected to a handle. The shortened profile of the syringe with the second plunger  123  removed (or the handle removed, in case of a plunger having a removable handle) fits into commonly used centrifuges. 
     The second step is to connect to the syringe  114  containing the specimen (e.g., fluid tissue), after the second plunger has been removed, a small micron vented luer cap  178  and then place the syringe inside the syringe tube holder  174 . The holder  174  has a solid bottom and also has an O-ring  176  attached to the solid bottom ( FIG.  10 A ). The O-ring lines up with the seal the plunger  113  makes with the outer wall  115  of the syringe barrel. Under high g-force, this O-ring serves to prevent any leaking from the syringe into the syringe tube holder  174 . G-force from the centrifuge causes the plunger to press against the O-ring on the bottom of the syringe tube holder. 
     Inside of the syringe  114  is a funnel shaped insert  116  (also referred to herein as a funnel) with a hole in the center. The density of the funnel is such that after density separation, target cells from blood or marrow will reside inside of the funnel. Consequently, after density separation of blood or marrow, plasma will reside at the top of the syringe  114  nearest the luer tip (container port)  130 , the target cells will reside inside of the funnel  116 , and red blood cells will reside beneath the funnel nearest the plunger  113 . Two example funnels  116   a ,  116   b  are shown in  FIG.  12   . 
     With respect to the materials used to make the funnel  116  and the shape of the funnel, it should be noted that various materials and shapes can work. When selecting a material, one consideration is whether the funnel  116  is to be molded or machined. In  FIG.  12   , the funnel  116   a  to the left is made of REXOLITE. This material has a density of 1.05 and is easy to machine. Alternatively, a plastic material may be used, such as ABS by Dow Chemical (part # 3105  FP EP) that has a density of 1.05 and is ideal for molding applications. A material that has a lower density than desired can have its density increased by adding screws or other material to the body of the funnel, as described above in reference to insert  16  of  FIG.  9   . Thus, various materials and plastics can be combined to fine tune the density of the funnel. For example, the funnel may be made of two parts or two different plastics. With respect to the shape of the funnel, many different shaped funnels will work. For example, a deeper, taller funnel, such as funnel  116   b  pictured to the right in  FIG.  12   , or a shallow, shorter funnel, such as the clear REXOLITE funnel  116   a  illustrated to the left, can be used in embodiments of the invention. Additionally, a bowl-shaped funnel or a funnel that has an inflection point, such that the angle of the wall is steeper at the bottom compared to the top, are all possible funnel shaped inserts that can be used in embodiments of the invention. Regardless of the shape, the funnel  116  has an upper funnel shaped portion  50  and a through hole  52  at the bottom as illustrated with respect to insert  16  ( FIG.  9   ). 
     The third step is to take the syringe  114  and tube holder  174  from the centrifuge, place the syringe inside the clamping mechanism  112  and engage the clamp as illustrated in  FIG.  13   . The pressure from the clamp will pinch the walls  115  of the syringe  114  so that the inner wall of the syringe barrel presses against the funnel  116 . This pressure will freeze (i.e., lock) the funnel in place. As shown, the clamping mechanism can include a lever  26  and cam  27  similar to the clamping mechanism described above in reference to  FIGS.  5 ,  7  and  8   . 
     The fourth step is to remove the vented cap  178  from the collection syringe  114  and connect to the collection syringe, via the upper luer connection  130 , the 30 ml plasma extraction syringe  120  connected to cannula  170  via manifold  160 , as shown in  FIG.  14 A . Cannula  170  fits through an upper injection port  162  connected to manifold  160  as shown in  FIG.  14 B . Also connected to manifold  162  are a side air vent  164  and a lower luer connection  166 . Thus, the collection syringe  114  is not vented during loading of the specimen but is vented, using manifold  160 , during extraction of the plasma and the target fraction inside the funnel. 
     After connection the plasma extraction syringe  120  and cannula  170  to the collection syringe  114 , the user pushes the extraction cannula  170  into the collection syringe. The cannula will advance until it hits the funnel  116  that has been frozen in place by the clamping mechanism  112 . As illustrated in  FIG.  15   , cannula  170  has a blunt closed end  171  and a hole  180  a certain distance above along its shaft. This hole is positioned to be high enough to extract only the plasma above but not the conents of the funnel  116 . Once the plasma is removed, the plasma extraction cannula  170  is removed from the collection syringe  114  and the plasma extraction syringe  120  is disconnected from the manifold  160 . Optionally, the syringe  120  and the manifold  160  are disconnected from the luer fitting  130  of the collection syringe and removed. 
     Once plasma is removed from container  114 , the same procedure as described above for syringe  120  and cannula  170  is repeated with the 5 ml concentrate extraction syringe  122  and cannula  172 .  FIG.  16    illustrates cannula  172  positioned against funnel (insert)  116 , the cannula including a side port  182  displaced from the distal end  173  of the cannula to withdraw fluid at a predetermined height above the distal end, e.g., within the funnel  116 , to withdraw substantially all of the target fraction. Extraction cannula  172  works the same way as the 30 ml extraction cannula except that hole (side port)  182  positioned near the cannula&#39;s blunt distal end  173  is close to the bottom of the funnel  116  so that the contents of the funnel are removed through hole  182  when vacuum pressure is applied via syringe  122 . Optionally, jet flushing, described above in reference to cannula  24  of  FIG.  9   , may be employed by ejecting fluid through hole  182  into the funnel  116  to release cells that adhere to the funnel inner surface. 
     Thus, in the examples illustrated in  FIGS.  15  and  16   , each cannula  170 ,  172  includes a closed end  171 ,  173  to close the hole  52  in the insert  116  and at least one side port  180 ,  182  to withdraw a component of the fluid, be it the selected component, e.g., buffy coat, or a component other than the selected component, e.g., plasma. Each cannula  170 ,  172  and the insert  116  may form a seal when the closed end of the cannula closes off the hole in the insert. 
     Additional features of the above embodiment, as illustrated in  FIGS.  10 A- 16   , are as follows: 
     a) The collection syringe (container)  114  is centrifuged ‘luer tip up’ and an O-ring or gasket is used to keep the syringe liquid-tight during centrifugation. If a syringe is centrifuged with the luer tip facing up, no cap or a vented cap for the luer tip should be used; otherwise the syringe distorts and leaks. 
     b) The use of luer connectors, injection ports, vented caps and a manifold  160  as a means to both 1) vent the collection syringe (container) and 2) insert a cannula into the syringe through the luer tip to extract fluid. 
     The collection syringe  114  is vented and fluid removed by employing the following novel features (see, e.g.,  FIGS.  14 A- 14 B ): 
     a) The connection between the extraction cannulas  120 ,  122  and the extraction syringes  170 ,  172  is air tight. 
     b) The seal around the upper injection port  162  and the cannula ( 170 ,  172 ) that has pierced it is also air tight. 
     c) The column of the manifold  160  is air tight with the exception of the air vent  164  at right angle to the syringes. 
     d) The connection  166  to the collection syringe  114  is air tight. 
     The collection syringe  114  is vented during the retrieval of the one or more target fractions. A target fraction is removed via a cannula using the negative pressure of an extraction syringe. The air vent  164  that used to accomplish this is not part of either syringe but is connected to both syringes, e.g., via the manifold  160 . The luer tip  130  of the collection syringe  114  is used as the extraction port. 
     An alternative extraction process using a syringe PRP (Platelet Rich Plasma) system will be described. In the embodiment described above, e.g., in reference to  FIG.  13   , the third step in the separation procedure is to take the syringe  114  and tube holder  174  from the centrifuge and place it inside of the clamping mechanism  112  to engage the clamp and freeze the funnel  116  in place. In the alternative embodiment described below, the clamping mechanism is not used. 
     An alternative to removing the plasma with a cannula after clamping the funnel  116  in place is to attatch a syringe  120  to the upper luer  130  of collection syringe  114  using a standard fem/fem luer connection and remove the plasma directly, without a cannula, by using the vacuum pressure of the two connected syringes ( FIG.  17   ). Since a standard luer connection is contemplated, the plasma can be removed under vacuum pressure by pulling back on the plunger of the plasma extraction syringe  120 . Thus, as the plasma is removed, the plunger  113  rises and the funnel  116  inside the barrel of the collection syringe  114  also rises. It is contemplated that the user removes the plasma until the funnel  116  reaches the top of the syringe  114 . After retrieving the plasma this way, the next step is to remove the plasma extraction syringe  120  from the collection syringe  114  and connect to the collection syringe, via the upper luer connection  130 , a vented 5 ml PRP extraction syringe, e.g., syringe  172  coupled to manifold  160 . 
     The target cells will not re-mix because the walls of the funnel  116  prevent fluid turbulance or interference from the inner wall of the syringe barrel. In addition, the center hole of the funnel  116  is sized such that at  1 G force, surface tension prevents fluid passage from below the funnel into the funnel. The PRP extraction syringe  122  is connected to a cannula  172  that fits through an upper injection port  162 , connected to a manifold  116  that has a side air vent  164  and a lower luer connection  166 . The cannula  172  can be shorter for this extraction process as the float  116  is at the top of the collection syringe  114 . Thus, the collection syringe  114  is not vented during loading of the specimen but is vented during extraction the target fraction inside the funnel. 
     After removal of the plasma and after the PRP extraction syringe  122  and cannula  172  are connected to the collection syringe  114 , the user pushes the extraction cannula  172  into the collection syringe. Since the insert  116  ends at the top of the syringe in this embodiment, the cannula  172  can be a set length, such that the cannula will advance the proper distance until it is approximately at the bottom of the funnel  116 . As described above in reference to  FIG.  16   , this cannula has a blunt closed end to butt against the funnel  116 , and a hole (e.g., a port) positioned a certain distance above the blunt end. Typically, the hole is positioned such that it ends up close to the bottom of the funnel when the blunt end of the cannula butts against the funnel. This ensures that the contents of the funnel  116  can be removed through the cannula  172  with the PRP extraction syringe  122 . 
     Returning to  FIGS.  1  and  2   , these figures illustrate embodiments described in previously filed application PCT/US2010/036696, which are useful to separate cells of a different fraction of a physiological fluid using centrifugation. The embodiments each include a collection tube or container that contains a funnel (e.g., an insert or float having a funnel-shaped portion) with a through hole in the funnel. The tube is designed to accept fluid, specifically blood or marrow. The funnel has a density such that after centrifugation, cells are captured inside the funnel. Two of the methods described for removing the cells contained inside the funnel after centrifugation are summarized below. 
     Method 1: The First Method Involves the Following Procedures: 
     a) After centrifugation, pinch the funnel in place by applying a clamp on the outside of the tube (e.g., container having a fixed bottom). The pressure on the tube causes the tube to flex. This then causes the inside wall of the tube to pinch against the outside wall of the funnel. 
     b) Once the funnel is secured in place, a double needle apparatus (inner cannula within outer cannula) is inserted through a center port. The blunt tip of the needle mates with the center hole of the funnel blocking off fluid below the funnel from fluid above the funnel. 
     c) The upper access hole (side port) of the double needle extracts all fluid that resides above the top of the funnel 
     d) The lower access hole (side port) of the double needle extracts all fluid that resides inside the funnel. 
     Method 2: The Second Method Involves the Following Procedure: 
     a) The fluid is loaded into a syringe (e.g., a container having a movable bottom or plunger) containing the funnel; the syringe can have no plunger handle or can have a removable plunger handle 
     b) The syringe has a center luer connection and a side luer connection, both of which are closed. 
     c) In one example, the center luer connection on the underside, inside the barrel of the syringe, has connected to it a blunt needle with side ports 
     d) After centrifugation, the target cells reside inside the funnel 
     e) Upper fluid (plasma) is removed via a plasma-syringe through the side luer connection. Because the system is a syringe, it is under vacuum pressure; consequently, as fluid is removed, the funnel and plunger move up. 
     f) Once the majority of the fluid has been removed, the blunt end of the needle meets the center hole of the rising float (funnel) and effectively seals all fluid above the float from fluid below the float. The port retrieving the plasma also mates with the float simultaneously so that no further fluid can be removed from the side port once the blunt needle impales the rising float. 
     g) The plasma syringe is removed and a vented cap added which now makes the system not under vacuum pressure. 
     h) Another syringe is connected to the center port and the target cells are removed. 
     Thus, of the two methods for removing the target cells from the funnel described above, the first involves freezing (e.g., clamping) the float in place and moving the needle assembly into the funnel to get the cells, while the second method involves using the vacuum pressure from the syringe to move the funnel up so that it impales itself into the fixed blunt needle assembly under the cap of the syringe. Please refer to WO 2010/138895 A2, e.g., FIGS. 13-33 and associated text, for a more complete description of the two methods described above. 
     Described below in reference to  FIGS.  18  and  19    is yet another apparatus and associated method for extracting cells from the collection funnel of a separation system. The apparatus and method include an upper and a lower funnel that are disposed inside the collection tube (container). As illustrated in  FIG.  18   , blood is loaded into a tube (container)  214  that defines a cavity that serves as the collection chamber for fluid. The tube  214  has an injection port  228  and an air vent  229 . Inside the collection tube  214  and in fluid communication with the injection port  228  is a flexible, optionally, clear tube  205  that is attached to a funnel  210  (referred to as the first or upper funnel) also disposed in the container  214 . The density of the upper funnel  210  is less than blood plasma. For example, the density of the upper funnel can be about 1.0. 
     After centrifugation, the bulk of the upper funnel  210  is floating above the plasma  2000 , as illustrated in  FIG.  19   . The bottom point of the upper funnel  210  has sunk slightly into the plasma or is in contact with the plasma. As shown, the funnel  210  is narrower than the diameter of the collection tube  214 . The funnel  210  has stabilization fins  215  at the top that extend from the funnel to approximately the sidewall of the tube  214  without touching the tube. These fins  215  are placed in such a manner that the upper funnel  210  remains oriented vertically with respect to the collection tube. The flexible tube  205  is attached at one end to the portion of the upper funnel where through hole  220  is located in the funnel. The other end of the tube  205  is connected to the port  228  in the top (e.g., cap) of the collection tube  214 . Tube  205  can be a small diameter clear tube that can flex and curl in on itself. Fluid is added to the collection tube  214  through the port  228  with a syringe. The fluid then travels through the length of flexible tube  205  and exits through hole  220  in the upper funnel  210  into the hollow chamber of collection tube  214 . Fluid is removed from the chamber through the same path. 
     Beneath the upper funnel  210  is a second funnel  216  that has a density of about 1.06, which is a higher density than the upper funnel and which allows the lower funnel  216  to float at the intermediate zone between red cells  2004  and plasma  2000 . After centrifugation, most of the platelets and white cells from blood or marrow reside within the lower funnel, as illustrated in  FIG.  19   . The lower funnel  216  has a through hole  52 , similar to other funnel shaped inserts described herein. Because the funnel  216  is open at the top and the bottom, it results in cleaner fluid flow and cleaner separation of components of the fluid, e.g., red cells, plasma, and target cells in the intermediate layer  2002  between red cells and plasma. 
     After centrifugation, the user can attach a syringe to the upper port  228  and begin to retrieve plasma first. The upper funnel  210  sinks as fluid in the chamber of tube  214  is removed but initially remains floating on the top of the fluid. After the desired amount of plasma has been removed, the user can switch syringes and begin to remove the remaining fluid above the lower funnel  216  and the contents of what is inside the lower funnel. 
     As additional fluid is removed, the upper funnel  210  continues to sink until it hits the lower funnel  216 . The angle of the upper funnel  210  is steeper than the angle of the lower funnel  216  so that the upper funnel fits into the lower funnel. The upper funnel  210  can have bottom stabilization fins  225 , so that the point of the upper funnel stops a certain distance below the bottom of the lower funnel  216 . This can also be accomplished by making the upper funnel  210  a certain height so that the upper stabilization fins  215  contact the upper surface of the lower funnel  216 . Additionally, a clamp  212  can be added after centrifugation that pinches the sidewall of the collection tube, such that the sidewall deforms and pinches against an outer surface of the lower funnel. In this way, the lower funnel  216  can be clamped in place and does not move as the upper funnel  210  mates with it during extraction of fluid. This allows the user to only withdraw the contents of the lower funnel  216  but not any fluid which is contained beneath the lower funnel. 
     As illustrated in  FIG.  19   , after centrifugation, red cells  2004  are below the lower funnel  216 , target cells of the density desired (e.g., huffy coat)  2002  are inside the lower funnel, plasma  2000  is above the target cells, and the upper funnel  210  is floating on top of the plasma. As described above, the user first removes plasma  2000  by attaching a syringe (not shown) to the center port  228  ( FIG.  18   ) of tube  214  and pulling back on the plunger of the syringe. This causes fluid to flow through the hole (port)  220  at the bottom of the upper funnel  210 , through the flex tube  205  and into the syringe. The user can remove as much plasma as is desired. Once the desired amount of plasma is removed, the user can remove the syringe containing the plasma and attach a second syringe (not shown) and remove the remaining fluid that is contained above and inside the lower funnel  216 . The stabilization fins  215 ,  225  and the relative height of the two funnels  210 ,  216  can be adjusted such that the upper funnel  210  dead ends against the lower funnel  216  so that no fluid from beneath the lower funnel is removed during the extraction process. 
     The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. 
     While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. For example, the double plunger collection syringe described herein may be used to separate fluid components other than those described herein and may be used in applications other than those described herein. Further, the example manifold disclosed herein, including the luer connection ports and micron vent, may be used to transfer fluids in a sterile manner in other applications, and may be used with syringes, cannulas, and containers other than those described herein. Further, inserts other than those illustrated and described herein may be used in combination with containers, cannulas and syringes to separate components of a fluid. For example, inserts need not have a density as described herein.