Abstract:
The invention relates to a system, and a set ( 21 ) of containers ( 23, 29, 31, 39, 43 ) and tubing ( 53, 57, 65 ) for use in such a system, for use in a centrifuge for separating components in fluid. The fluid is moved from container to container during centrifugation by pistons ( 27, 33, 35, 47 ) provided in the containers. The ratio of mass divided by the cross-sectional area of the container that each piston moves in is different for each piston ( 27, 33, 35, 47 ). During centrifugation fluids can be moved from a container having a piston with a high mass to cross-sectional area ratio to a container having a piston with a lower mass to cross-sectional area ratio.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a filing under 35 U.S.C. § 371 and claims priority to international patent application number PCT/SE2007/000388 filed Apr. 23, 2007, published on Nov. 8, 2007, as WO 2007/126357, which claims priority to patent application number 0608451.1 filed in Great Britain on Apr. 28, 2006. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to systems and sets of the types mentioned in the preambles of the independent claims for performing the separation of different density components in fluids 
       BACKGROUND OF THE INVENTION 
       [0003]    Some fluids, in particular biological fluids such as blood, contain a number of valuable and/or medically or pharmaceutically useful components. Much effort has been expended in finding methods and devices which can efficiently separate and collect such valuable/useful components in relatively pure concentrations. One such way for separating components out of a sample of blood is known from U.S. Pat. No. 6,733,433. This patent describes a system for centrifuging biological fluid in which a sample is placed inside a variable volume cylindrical chamber provided with an axially movable piston. The cylinder is spun rapidly around its longitudinal axis which causes the components in the biological fluid to separate into fractions arranged in concentric rings with the densest fraction nearest the circumference of the chamber and the least dense fraction in the centre of the chamber. The different fraction can be emptied in turn from the chamber though a central opening in the top of the chamber by using compressed air to move the piston towards the top of the chamber. The central opening is connected by a rotary seal to a system of valves, tubes and collection bags to which the different fractions can be directed. An optical sensor on a tube leading from the central opening measures the light absorbance in the tube and the changes in the signal from the optical detector are used by a control device to determine when different fractions pass the optical sensor and to control the valves so that the fractions are directed to the correct collection bag. 
       SUMMARY OF THE INVENTION 
       [0004]    According to the present invention, systems and sets are provided for separating fluids, particularly biological fluids, into fractions by means of a system having the features present in the characterizing part of claim  1 , and a set having the features mentioned in the characterizing part of claim  6 . 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0005]      FIG. 1  shows a schematic plan view of a first embodiment of a centrifuge in accordance with the present invention. 
           [0006]      FIG. 2  shows schematically a first embodiment of a disposable set for separating and collecting fractions of biological fluids in accordance with the present invention arranged in a centrifugal chamber. 
           [0007]      FIG. 3  shows steps in an embodiment of a method for separating and collecting fractions of blood using the disposable set of  FIG. 2 . 
           [0008]      FIG. 4  shows steps in an embodiment of a method for separating and collecting fractions of blood using the disposable set of  FIG. 2 . 
           [0009]      FIG. 5  shows steps in an embodiment of a method for separating and collecting fractions of blood using the disposable set of  FIG. 2 . 
           [0010]      FIG. 6  shows steps in an embodiment of a method for separating and collecting fractions of blood using the disposable set of  FIG. 2 . 
           [0011]      FIG. 7  shows steps in an embodiment of a method for separating and collecting fractions of blood using the disposable set of  FIG. 2 . 
           [0012]      FIG. 8  shows steps in an embodiment of a method for separating and collecting fractions of blood using the disposable set of  FIG. 2 . 
           [0013]      FIG. 9  shows steps in an embodiment of a method for separating and collecting fractions of blood using the disposable set of  FIG. 2 . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0014]      FIG. 1  shows schematically a plan view of a centrifuge  1  in accordance with the present invention in which features well-known to the skilled person have been omitted. Centrifuge has a body  3  which supports a central rotatable shaft  5  to which a plurality, in this case  4 , of centrifugal chamber supporting arms  7  are attached. Each arm supports its own centrifugal chamber  9 . A imaging device  11 , for example a video camera  13  and a high speed camera flash  15  are arranged to image part of the path that a centrifugal chamber  9  follows when being rotated around the central shaft  5  and each centrifugal chamber  9  has transparent wall or window  17  facing towards the imaging device  11 . Centrifuge  1  is controllable by a control device  19  such as a microprocessor or a computer which controls the speed of rotation of the centrifuge as well as controlling the flash  15  and valves described below and controlling and processing images from imaging device  11 . Flash  15  is controlled so that it illuminates each centrifugal chamber as it passes the same position in the field of view of camera  13  in order to achieve a pseudo-stationary or “still” image of each chamber  9 . The connection between the control device  19  and centrifuge  1 , valves, imaging device  11  and flash  15  can be through wires and/or it can be wireless, for example by infra red or radio frequency communication. 
         [0015]      FIG. 2  shows schematically a centrifugal chamber  9  and a set  21  of containers and tubing for separating and collecting components of a biological fluid. Set  21  comprises: a biological fluid-receiving container  23  for receiving the biological fluid to be separated, said container  23  having a cylindrical wall  25  and a bore of cross-sectional area AF containing a movable piston  27  of mass MF in sealing contact with said cylindrical wall  25 , the volume of the bore between the piston  27  and the base  55  of container  23  forming a biological fluid receiving cavity  28 ; a first separated-fraction-receiving container  29  for receiving a separated fraction, said container  29  preferably being in the form of a flexible bag or cylinder of variable volume; an optional separated-fraction-cleaning cylinder  31  having a cylindrical wall  33  and a bore of cross-sectional area AS containing a movable piston  35  of mass MS in sealing contact with said cylindrical wall, the volume of the bore between the piston  35  and the base  32  of container  31  forming a cavity  34  of sufficient volume for receiving a separated fraction and wash buffer; an optional wash buffer containing container  37  having a cylindrical wall  39  and a bore of cross-sectional area AB containing a movable piston  41  of mass MB in sealing contact with said cylindrical wall  39 , the volume of the bore between the piston  41  and the base  38  of container  37  forming a cavity  40  for containing a wash buffer; a first additive-containing container  43  having a cylindrical wall  45  and a bore of cross-sectional area AA containing a movable piston  47  of mass MA in sealing contact with said cylindrical wall, the volume of the bore between the piston  47  and the base  44  of container  43  forming a cavity  48  for storing an additive solution; and a second separated-fraction-receiving container  49 . Optionally a second additive-containing container  51  may be included in the set arranged so that additive leaving first additive-containing container  43  enters into second additive-containing container  51  through an inlet  50  at or near one end thereof, mixes with the contents of second additive-containing container  51 , the mixing being optionally assisted by a mixer  52 , and the mixed additive leaves second additive-containing container by an outlet  54  placed at or near the opposite end thereof. 
         [0016]    Set  21  further comprises a first, preferably flexible, tubing  53  which connects the base  44  of first additive-containing container  43 , (via second additive-containing container  51  if fitted) to the base  55  of fluid-receiving container  23 , a second, preferably flexible, tubing  57  which leads from a passage  59  through piston  27  to the leg of a T-junction  61  which leads via a first arm and third, preferably flexible, tubing  63  to second separated-fraction-receiving container  49  and via the second arm and fourth, preferably flexible, tubing  65  to separated-fraction-receiving container  29 . If optional separated-fraction-cleaning cylinder  31  and optional wash buffer containing cylinder  37  are presented then their bases  32 , respectively  38 , are connected to tubing  65  by tubing spur  67  and tubing spur  69  respectively. 
         [0017]    Centrifugal chamber  9  has a cavity  71  with preformed depressions  73  adapted to receive and hold in place set  21  in cavity  71  in a predetermined orientation in which the bases of containers  23 ,  31 ,  37 ,  43  are further away from the centre of rotation of the centrifuge than their opposite ends. A first remote controllable valve  75  is positioned to be able to allow or prevent fluid flow through tubing  53 . A second remote controllable valve  77  is positioned to be able to allow or prevent fluid flow through tubing  57 . A third remote controllable valve  79  is positioned to be able to allow or prevent fluid flow through tubing  63 . A fourth remote controllable valve  81  is positioned to be able to allow or prevent fluid flow through tubing  65 . A fifth remote controllable valve  83  may be positioned to allow or prevent fluid flow through tubing spur  67 . A sixth remote controllable valve  85  may be positioned to allow or prevent fluid flow through tubing spur  69 . 
         [0018]    In order to allow a volume of biological fluid to be easily introduced into biological fluid-receiving container  23  an inlet line  87  with a preferably sealable inlet port  89  may be provided at any suitable position on the set  21 , for example between valve  75  and the biological fluid-receiving container  23  or between valve  77  and the biological fluid-receiving container  23 . 
         [0019]      FIGS. 3-9  show steps in an embodiment of a method in accordance with the present invention for separating a predetermined component from a sample of biological fluid using density gradient media  91  as an additive and a wash buffer  93  for washing the predetermined component. In this example the biological fluid is blood  95  and the density of the density gradient media is chosen to be higher than that of mononucleotide cells and less than that of red blood cells in the blood. The first and second additive containers  43 ,  51  are filled with density gradient media  91  and wash buffer containing container  37  is filled with wash buffer  93 .  FIG. 3  shows the step of filing biological fluid receiving container  23  with the sample of blood  95  via inlet port  89  and inlet line  87 . This is achieved by positioning piston  27  at the base of container  23 , closing valves  75  and  77  and injecting the blood sample  95  through inlet port  89  into inlet line  87 . The closed valves  75  and  77  prevent the blood sample from entering tubing  53  and  57 , which leads to piston  27  being pushed up away from the base  55  of container  23  as blood sample  95  is introduced. The introduction of blood sample  95  into container  23  can take place when the set  21  is inside a centrifugal chamber  9  or more preferably, before being placed into centrifugal chamber  9 . 
         [0020]    Once blood sample  95  has been introduced and set  21  positioned into centrifugal chamber  9  if it was not already there, centrifugation of the set  21  is begun. The control device causes shaft to rotate at the desired speed and then the density gradient media  91  is added to sample  95 . This is achieved by opening valve  75 . No pump is need to move the density gradient media  91  from additive container  43  to container  23 —this because the ratio of the mass of movable piston  47  over the cross-sectional surface area of container  43  is greater than ratio of the mass of piston  27  over the cross-sectional area of container  23  which cause a pressure differential between the containers which forces the density gradient media  91  into container  23 . This flow of density gradient media continues until the pressure differential is equalized or valve  75  is closed. As the centrifugation continues after the flow of density gradient media has ceased the components of the blood sample and the density media move to levels in container  23  which are dependent on their densities and form distinct layers as shown in  FIG. 4 . In this example, blood sample  95  has been separated in a layer of dense red blood cells  101 , above which is a layer of density gradient media  91 . A layer of mononucleotide cells  103  lies above the density gradient media  91  and a further layer of less dense blood plasma  105  lies above the layer of mononucleotide cells  103 . 
         [0021]    Control device  19  can be provided with software for image processing and by processing the picture signal from imaging device  11  it can identify when the components have separated into substantially stable layers. Once substantially stable layers have been identified (or after a predetermined time since the start of centrifugation has elapsed), control device  19  commands valves  77  and  79  to open. This allows piston  27  to move towards the base of container  23  under the force Acc generated by the rotation of centrifugal chamber  9  which causes the fluid in container  23  nearest to piston  27 , blood plasma  105 , to leave the container  23  via passage  59 . The blood plasma  105  passes along tubing  57 , though valve  77 , along tubing  63  to second separated-fraction-receiving container  49 . Once all the blood plasma  105  has left container  23 , the mononucleotide cells  103  start to leave container  23  via passage  59  as shown in  FIG. 5 . 
         [0022]    Control device  19  is preferably provided with software which can calculate the rate of flow of blood plasma through tubing  57  by measuring the speed of displacement of piston  27  and using the known volume per unit length of tubing  57 . It can calculate when the last of the blood plasma  105  and the first of the mononucleotide cells  103  will reach T-junction  61  and can command valve  79  to close and valve  83  to open at this time (or shortly before this time to ensure the maximum yield of mononucleotide cells  103  by avoiding the risk that some mononucleotide cells  103  enter tubing  63 ). 
         [0023]    In this embodiment of a method according to the present invention it is desired to clean the mononucleotide cells  103  before collecting in separated-fraction-receiving container  29  then once valve  79  is closed valve  81  is kept closed and the valve  83  is opened. This causes the mononucleotide cells  103  to flow into separated-fraction-cleaning container  31  via tubing  65  and tubing spur  67  as shown in  FIG. 6 . As the ratio of piston mass over container cross-sectional area of container  31  is less than that of container  23 , this flow occurs without the aid of any pump. Control device  19  is preferably provided with software which can calculate the rate of flow of density gradient media  91  through tubing  57  by measuring the speed of displacement of piston  27  and using the known volume per unit length of tubing  57 . It can calculate the time when the last of the mononucleotide cells  103  and the first of the density gradient media  91  will reach valve tubing spur  67  and can command valves  77  and  83  to close at this time (or shortly before this time) to prevent any density gradient media  91  passing into tubing spur  67  into separated-fraction-cleaning container  31 . 
         [0024]    The mononucleotide cells  103  can be cleaned in separated-fraction-cleaning container  31  by opening fifth remote controllable valve  83  and sixth remote controllable valve  85 . As the ratio of the mass of movable piston  41  over the cross-sectional surface area of container  37  is greater than ratio of the mass of piston  35  over the cross-sectional area of container  31  a pressure differential is formed between the containers  31 ,  37  which forces the wash buffer  93  from container  37  into container  31  until an equilibrium is reached. Wash buffer  93  is preferably selected to have a specific density which is less than that of the mononucleotide blood cells  103 . This flow of wash buffer  93  into container  31  lifts the mononucleotide cells  103  from the base  32  of container  31  and if the speed of the incoming wash buffer  93  is sufficiently high it suspends them in the flow of incoming wash buffer  93  at a distance from the base  32 . As shown in  FIG. 7  they remain suspended as incoming wash buffer  93  flows through the layer of mononucleotide cells  103 , thereby washing them in a process called “elutriation”. After a predetermined time or after a predetermined volume of wash buffer  93  has entered container  31  or once piston  35  has reached a predetermined position, valves  83  and  85  are closed. This allows the mononucleotide cells  103  to collect at the base of container  31  as shown in  FIG. 8 . 
         [0025]    The mononucleotide cells  103  can be transferred to first separated-fraction-receiving container  29  by opening valves  81  and  83 . This allows the force exerted by piston  35  on the contents of container  31  to push the mononucleotide cells  103  through tubing spur  67  and tubing  65  via valves  81  and  83  into first separated-fraction-receiving container  29  as shown in  FIG. 9 . Control device  19  is preferably provided with software which can calculate the rate of flow of mononucleotide cells  103  through tubing  65  and  67  by measuring the speed of displacement of piston  35  and using the known volume per unit length of tubing  65  and  67 . It can calculate the time when the last of the mononucleotide cells  103  and the first of the wash buffer  93  will reach valve  81  and can command valves  81  and  83  to close at this time (or shortly before this time) to prevent any wash buffer  93  from valve  81  and into separated-fraction-receiving container  29 . The centrifuge can then be stopped and the set  21  remove from the centrifuge chamber  9  for further processing. 
         [0026]    In a second embodiment of a method in accordance with the present invention it is desired to collect the mononucleotide cells  103  without cleaning. This second embodiment of a method is the same as the first embodiment of a method in accordance with the present invention (except that it is no longer necessary to provide wash buffer in container wash buffer containing container  37 ) up to the point when blood plasma is contained in second fraction container  49  and valve  79  has been closed. Once valve  79  is closed, in the second embodiment of a method in accordance with the present invention valve  81  is opened and the mononucleotide cells  103  flow though tubing  65  into first separated-fraction-receiving container  29  due to the force that piston  27  exerts on the contents of container  23 . Once all the mononucleotide cells  103  have left container  23 , the density gradient media  91  starts to leave container  23  via passage  59 . Control device  19  is preferably provided with software which can calculate the rate of flow of density gradient media  91  through tubing  57  by measuring the speed of displacement of piston  27  and using the known volume per unit length of tubing  57 . It can calculate the time when the last of the mononucleotide cells  103  and the first of the density gradient media  91  will reach valve  81  and can command valve  81  to close at this time (or shortly before this time) to prevent any density gradient media  91  passing through valve  81  into first separated-fraction-receiving container  29 . The centrifuge can then be stopped and the set  21  remove from the centrifuge chamber  9  for further processing. 
         [0027]    In the embodiments of the present invention described above only one type of density gradient media was employed and this formed a layer between components of the biological sample having a density greater than the density gradient media and components having a density less than the density gradient media. Often it is desirable to separate a biological fluid into more than two fractions separated by a layer of density gradient media and this requires the use of more than one density of density gradient media. Preferably this is achieved by mixing together, in varying proportions two density gradient media having different densities—the denser having a original density A and the less dense having a density B—either to form a substantially continuous gradient of gradient density media (where the density ranges from A to B) or, by mixing predetermined proportions of each of the original density gradient media (e.g. 10% A plus 90% B, 50% A plus 50% B, etc), to achieve a number of intermediate-density density gradient media having densities lying between the density of the denser original gradient density medium A and the least dense original density gradient medium B. 
         [0028]    In a third embodiment of a method in accordance with the present invention, it is desired to use a continuous gradient of density gradient media. This method differs from the methods of the first and second embodiments of the previous invention by starting with a first, preferably densest gradient density medium A in first additive container  43  and a second least dense gradient density medium B in second additive container  51 . A gradient of density gradient media is achieved by actuating mixer  52  and opening valve  73 . Opening valve  73  allows density gradient medium A to flow from first additive container  43  into second additive container  51  where it is mixed by mixer  52  with density gradient medium B to form a intermediate-density density gradient medium with a density between A and B. As the intermediate-density density gradient medium leaves second additive container  51  and flows into biological fluid-receiving container  23  the proportion of density gradient medium A to density medium B in second additive container  51  increases and the density of the intermediate-density gradient density medium leaving second additive container  51  increases as well. This leads to a gradient of increasing density intermediate-density density gradient media being introduced into biological fluid-receiving container  23 . Once the desired volume of density gradient media has been introduced into biological fluid-receiving container  23  then mixer  52  is deactivated and valve  75  closed. The method then continues in a similar fashion to the method previously described, the main difference between the methods being that if the correct density gradient media gradient has been achieved then the target component(s) of the biological fluid will be separated into more layers, with a layer of density gradient media separating these layers. 
         [0029]    The above examples illustrate specific aspects of the present invention and are not intended to limit the scope thereof in any respect and should not be so construed. Those skilled in the art having the benefit of the teachings of the present invention as set forth above, can effect numerous modifications thereto. These modifications are to be construed as being encompassed within the scope of the present invention as set forth in the appended claims.