Abstract:
A separation device for separating a wanted end product from a liquid sample comprises a container ( 2 ) having a first end ( 5 ) and a second end ( 7 ), the first end having a central orifice ( 6 ), a plunger ( 3 ) slideably disposed in the container ( 2 ) to define a variable liquid receiving chamber between the plunger ( 3 ) and the orifice ( 6 ), and a permeable partition member ( 9 ) mounted to the plunger ( 3 ) in a spaced relationship thereto to define a compartment ( 12 ) between the partition member ( 9 ) and the plunger ( 3 ) for receiving liquid density gradient medium ( 13 ), wherein liquid may be drawn into the container ( 2 ) and expelled therefrom, respectively, through the orifice ( 6 ) by movement of the plunger ( 3 ) relative to the container ( 2 ). A method for separating a wanted end product from a liquid sample is also disclosed.

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/SE2009/050909 filed Jul. 20, 2009, published on Feb. 4, 2010 as WO 2010/014033, which claims priority to application number 0801746-9 filed in Sweden on Jul. 31, 2008. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to a separation device adapted for separation of a wanted end product from a sample by centrifugation, and to a method for separation of a wanted end product from a sample using the separation device. 
       BACKGROUND OF THE INVENTION 
       [0003]    The separation of cell containing samples, for example blood, into different fractions by using centrifugation and a density gradient medium has been practised for some time. The principle used is to provide for example a blood sample together with a density gradient medium in a tube and then put the tube into a centrifuge. The density gradient medium is suitably chosen such that after centrifugation red blood cells are collected at the bottom of the tube, below the density gradient medium, and the wanted fraction, for example mono nuclear cells, MNCs, will stay at the top of the density gradient medium. The plasma will also be separated and stay above the MNCs. In order to collect the MNCs a pipette is normally used. Typically, the pipette is manually lowered into the tube such that the open end of the pipette is provided in the MNC band. Thereafter the MNCs are manually drawn up through the pipette. This is a tricky process since only MNCs are wanted. The amount of density gradient medium and plasma should be minimised. Such a manual process using centrifugation and a density gradient medium is for example described in Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21, Suppl 97 (Paper IV), 77-89, 1968. 
         [0004]    A problem with this method is as described above that the manual handling of the pipette when collecting the MNCs is difficult. The yield and purity of the end product will differ due to variations in the collection. 
         [0005]    Another problem is related to the sample application. The sample needs to be applied very carefully on top of the density gradient medium in order not to be mixed with the density gradient medium before centrifugation. 
       SUMMARY OF THE INVENTION 
       [0006]    One object of the invention is to provide a separation device that is easy to use, including easy sample application and easy withdrawal of sample, where the wanted end product easily can be retrieved as pure as possible. 
         [0007]    Another object of the invention is to provide a separation device which has a simple design and construction and is suitable for mass production. 
         [0008]    These objects are achieved with a separation device according to claim  1  and with a method according to claim  11 . With this device and method it is easy to apply the sample and easy to retrieve the wanted end product. 
         [0009]    Suitable embodiments are described in the dependent claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0010]      FIG. 1  is a schematic cross-sectional view of a syringe device according to a first embodiment of the invention. 
           [0011]      FIGS. 2A and 2B  are schematic illustrations of different steps when using the syringe device in  FIG. 1  for separation of a sample. 
           [0012]      FIG. 3  is a schematic view of a syringe device according to a second embodiment of the invention. 
           [0013]      FIG. 4  is a schematic partial view of an embodiment of a collapsible partition member. 
           [0014]      FIG. 5  is a schematic illustration of different steps when using the syringe device in  FIG. 3  for separation of a sample. 
           [0015]      FIG. 6  is a schematic illustration of different steps when using the syringe device in  FIG. 3  for separation of a sample by means of a linear gradient of density gradient medium. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0016]    According to the invention, a syringe device and a method for the separation of a wanted end product from a sample is provided. 
         [0017]    The sample could be for example a body fluid such as blood or bone marrow, a body tissue, such as adipose tissue or a sample containing cell cultures or cell clusters or cell fragments such as organelles. The wanted end product could be cells of different kinds, such as for example stem cells, mononuclear cells (=MNCs), hematopoetic cells and progenitor cells or cell fragments/organelles such as for example mitochondria, golgie, endoplasmic reticulum and cell nuclei. 
         [0018]    A density gradient medium (density separation medium) is provided inside the syringe before sample is applied and the syringe is centrifuged. The term “density gradient medium” is to be interpreted in a broad sense herein. While the density gradient medium usually is a medium which may form a density gradient upon centrifugation or sedimentation, it may also be a medium which does not form a density gradient but merely has a different density than the sample medium and forms a step gradient with the sample medium. Density gradient media used for this type of separation, such as for example FICOLL™, PERCOLL™, sucrose, inorganic salt, e.g. caesium chloride, are well known in the art. The density of the medium should be chosen such that at least one fraction of the body fluid will be separated and positioned below the density gradient medium after centrifugation. In case, for example, blood is separated, the red blood cells should preferably be separated and positioned at the bottom of the device under the density gradient medium after centrifugation. 
         [0019]    Some density gradient media, like FICOLL™, for example, efficiently aggregate red blood cells at room temperature. When on centrifugation with such a medium, cells in a blood sample sediment towards and come in contact with the blood/density gradient medium interface, the red blood cells start to aggregate which increases the rate of sedimentation of the red cells. The red cells therefore rapidly collect as a pellet at the bottom of the syringe, where they are well separated from lymphocytes. Granulocytes will also sediment to the bottom of the density gradient medium layer, facilitated by the increase in their densities caused by contact with the slightly hypertonic gradient density medium. Thus, on completion of centrifugation, both granulocytes and red blood cells will be found at the bottom of the syringe, beneath the gradient density medium. Lymphocytes, monocytes and platelets, on the other hand, are not dense enough to penetrate into the gradient density medium layer. These cells will therefore collect as a concentrated band at the interface between the original blood sample and the gradient density medium layer. 
         [0020]    Alternatively, two or more different density gradient media may be used. If two density gradient media are used and blood is the sample that should be separated, the density gradient medium with lowest density can preferably be of such composition that the red blood cells are not caused to aggregate. Examples of such density gradient media are PERCOLL™ or sucrose. This is to prevent red blood cells from possibly enclosing wanted cells during the aggregation process and thereby decrease yield of the wanted end product. The density gradient medium with higher density can, however, be of such composition that aggregation of the red blood cells is induced, such as e.g. FICOLL™ as mentioned above. 
         [0021]    As still another alternative, a linear gradient produced by mixture of two different densities of a density gradient medium (or, optionally, two different density media) may be used, as will be described in more detail below. 
         [0022]    A characteristic feature of the syringe device of the invention is a partition member attached to the syringe plunger and spaced a predetermined distance thereto to define a gradient density medium compartment between the plunger and the partition member. The partition member should, on the one hand, prevent mixing of the gradient density medium with sample fluid applied on top of the partition member but, on the other hand, be permeable to permit passage of liquid and usually also of sample components, such as e.g. cells, therethrough by the application of a force, such as by centrifugation or other forced movement of the partition member relative to the liquid. 
         [0023]    The partition member may, for example, be passive like a filter, a grid, etc, preferably of capillary type, or active, such as e.g. a plate with densely packed microvalves. 
         [0024]    In one embodiment, the partition member is rigidly fixed to the plunger. In this case, if the sample is blood, for example, on completed centrifugation the desired MNC band will be on top of the density gradient medium slightly above the partition member (i.e. at the interface between sample and density gradient medium). 
         [0025]    In another (currently preferred) embodiment, the partition member is “collapsible” before or during centrifugation to permit displacement of the partition member towards the syringe plunger. Thereby, the distance between the partition member and the MNC band will be sufficiently increased to facilitate the harvesting or collection of the MNC band. 
         [0026]    Such a collapsible partition member, e.g. a capillary filter, may be accomplished in various ways. For instance, the attachment of the partition member to the syringe plunger may be designed to cause displacement of the partition member towards the plunger when affected by a sufficient centrifugal force during centrifugation. Alternatively, the attachment of the partition member may be designed to permit such displacement by, e.g., manual actuation of the plunger or partition member before centrifugation. In still another alternative, the attachment of the partition member may be designed to permit the plunger to be displaced towards the partition member. 
         [0027]    Collapsibility by manual actuation may be accomplished, for example, by the plunger and the partition member being attached to each other through two (or more) telescoping sliding or threadedly engaged cylinders or the like, whereby one cylinder may be partly or wholly pushed (optionally by centrifugal force) or screwed, respectively, into the other. Another example of collapsible attachment structure is a bellows type member which may be compressed on centrifugation and kept in the collapsed state by a suitable latch or friction means, for instance. In still another example, the partition member is mounted to the upper end of a shaft or bar member which sealingly and slidingly extends through the center of the syringe plunger, so that axial movement of the shaft or bar varies the distance between the plunger and the partition member. 
         [0028]    Embodiments of collapsible partition members will be described in more detail below. 
         [0029]    The desired cell band or bands may be removed from the syringe device and collected in various ways. Suitable collection means include, for example, flexible bags, vials, tubes, or other containers or receptacles. 
         [0030]    In one embodiment, collection of waste liquid (the plasma) as well as of the desired MNC band(s) takes place through flexible containers attached to the syringe inlet/outlet. After the sample has been introduced into the syringe, and optionally after centrifugation to cause cell banding, a first flexible container is attached to the syringe. The supernatant plasma fraction may then be displaced into the container by, for example, centrifugation at a higher speed or by manual or automatic displacement of the syringe plunger towards the syringe inlet/outlet in a dedicated holder or similar device. A second (smaller) container is then attached to the syringe to collect the desired MNC band (or bands) by manual or automatic operation of the syringe in the dedicated holder. The collected MNC band is then ready for further processing. 
         [0031]    Alternatively, the syringe contents may be displaced via an applied conduit, such as a tubing, into a number of fraction collection tubes or the like. The displacement through the tubing is then monitored visually or by a detection means, e.g. a photocell, so that various separated fractions may be collected in respective collection tubes. Such a process may, of course, also be automated. 
         [0032]    Optionally, an initial step of depletion of unwanted cells may precede the density separation. For example, the sample may be pre-incubated with beads containing immobilized affinity ligands specific to the unwanted cells. 
         [0033]    An automated or semi-automated separation procedure according to the present invention including such pre-treatment of the sample may comprise the following steps:
       Sample is pre-incubated with beads.   A syringe device containing density gradient medium is placed in a dedicated holder and sample is applied by actuation of the holder, such as by pushing a button.   The syringe device is capped and placed in a centrifuge, and centrifuged to separate the sample components.   The syringe device is then again placed in the dedicated holder and a tubing is connected, whereupon cell fractionation is started, such as by pushing a button.       
 
         [0038]    In case a linear density gradient is to be used, such a gradient may be formed by placing an empty syringe device in the holder and applying a varying mixture of high and low density medium, and then applying the sample. 
         [0039]    Embodiments of the present invention will now be described in more detail with reference to the accompanying drawings. 
       First Embodiment 
       [0040]    An embodiment of the syringe device of the present invention is illustrated in  FIGS. 1 and 2A ,  2 B. With specific reference to  FIG. 1 , the syringe device, generally designated by reference numeral  1 , includes a syringe cylinder  2  in which a plunger including a piston plug  3  with a piston rod  4  is slideably mounted. The syringe cylinder  2  has a tapering top end  5  (here a frustrated cone) with a sample inlet/outlet  6 , and an open bottom end  7  (“top” and “bottom” referring to the position of the syringe device  1  on the drawing). The syringe cylinder  2  is slideably mounted in a supporting cylinder or bucket  8 . A partition member  9  in the form of a filter or grid, for example, in the following for simplicity referred to as filter  9 , is rigidly attached to the piston plug  3  spaced thereto, e.g. by a cylindrical member  10 . In the Figure, the filter  9  is shown to be at its top position in the syringe cylinder  2  adjacent to the cone-shaped top end  5 , and with the bottom end of the piston rod  4  spaced from the bottom of the supporting bucket  8 . The filter  9  divides the volume between the piston plug  3  and the inlet/outlet  6  into a sample compartment  11  above the filter  9 , and a density gradient medium compartment  12  below the filter  9 . As is readily seen, the volume of the density gradient medium compartment  12  is constant, whereas the volume of the sample compartment  11  varies depending on the position of the piston plug  3  in the cylinder  2 . In the Figure, the compartment  12  is filled with a liquid density gradient medium  13 . The component parts of the syringe device  1  are made from a suitable material known to a person skilled in the art. 
         [0041]    The syringe device  1  is designed to be placed in the rotor of a centrifuge, either directly or through a suitable adapter. 
         [0042]    A method of using the syringe device in  FIG. 1  will now be described with reference to  FIGS. 2A and 2B . 
         [0043]    In  FIG. 2A , the different method steps are illustrated by subfigures A 1  to A 12 . 
         [0044]    A 1 : The syringe device  1  is ready for use with the filter  9  in its top position as shown in  FIG. 1 , i.e. with the piston rod  4  at a distance from the bottom end  8   a  of the supporting bucket  8 , and with the compartment  12  filled with density gradient medium  13 , e.g. FICOLL™, up to the top surface of filter  9 . 
         [0045]    A 2 : A sample applicator  20  with sample  21 , e.g. blood, is connected via a tip portion  22  to the inlet/outlet  6  of the syringe device  1 . Optionally, the sample has first been subjected to a pre-incubation with beads containing immobilized specific affinity ligands for depletion of unwanted cells. 
         [0046]    A 3 : Sample  21  is applied to the syringe device  1  by actuation of the sample applicator  20 . Initially, the introduced sample  21  forces the piston plug  3  downwards until the piston rod  4  contacts the bottom  8   a  of the supporting bucket  8 . During the whole sample application process, the filter  9  keeps the sample separated from the density gradient medium  13 . 
         [0047]    A 4 : Continued application of sample  21  then forces the syringe cylinder  2  to be displaced upwards relative to the piston plug  3  with the attached filter  9 . 
         [0048]    A 5 : When all sample  21  (or the desired amount of sample) has been introduced into the sample compartment  11 , the sample applicator  20  is removed. 
         [0049]    A 6 : A flexible waste container  23  is attached to the syringe inlet/outlet  6  to serve as a waste compartment. Preferably, the inlet of the flexible container  23  is constrained in the sense that a predetermined force or pressure is necessary to permit the entry of fluid into the container. 
         [0050]    A 7 : The syringe assembly is then placed in a centrifuge and centrifugation is started. During centrifugation at a selected first speed, the cells in the sample  21  are forced through the filter  9  and separated and banded in the density gradient medium  13 . When the sample is blood, for example, the red cells (which have a specific gravity higher than the selected density gradient medium) pass through the density gradient medium  13  and are consolidated in a layer  24  at the bottom of the density gradient medium compartment  12 , whereas the wanted fraction containing MNCs (which have a specific gravity less than the density gradient medium) is concentrated in a band  25  on top of the density gradient medium  13  above the filter  9  at the interface between the plasma  26  and the density gradient medium. 
         [0051]    A 8 : The centrifugal force is then increased by centrifugation at a second, higher speed which forces the syringe cylinder  2  to move downwards to eventually contact the bottom  8   a  of supporting bucket  8 , thereby displacing the plasma  26  into the flexible container  23 . 
         [0052]    Alternatively, the separation step in A 7  and the plasma displacement step in A 8  may be combined and done simultaneously by controlling the centrifugation force to fine-tune the speed of cells sedimentation and displacement of the plasma. 
         [0053]    As still another alternative, the displacement of the plasma may be done manually or automatically in a dedicated holder or apparatus similar to, or the same as that outlined with reference to subfigures A 9  to A 12  below. 
         [0054]    A 9 : The flexible container  23  with plasma  26  is then removed, and replaced by a (small) sample container  27 . 
         [0055]    A 10 : In the shown embodiment, the syringe device  1  is then put in a dedicated (specially designed) holder or apparatus, schematically illustrated at  28 , which has a part  28   a  capable of actuating the piston rod  4 , either by manual operation or automatically. 
         [0056]    A 11 : By actuation of the piston rod  4  in the holder  28 , here simply by pressing syringe device  1  downwards in the holder, the piston plug  3  is moved upwards in the syringe cylinder  2 , displacing the MNC band  25  into the sample container  27 . 
         [0057]    A 12 : When the displacement of the MNC band is completed, the sample container  27  containing the MNCs is removed and optionally capped. 
         [0058]    An alternative way of performing the cell banding centrifugation and displacement centrifugation described in the steps of subfigures A 6  to A 8  in  FIG. 2A  above is illustrated in  FIG. 2B  by subfigures B 1  to B 4  (wherein the same reference designations as in  FIG. 2A  are used for corresponding parts). 
         [0059]    B 1 : After the sample applicator  20  has been removed (subfigure A 5  in  FIG. 1 ), a stopper  29  is put on the syringe inlet/outlet  6 . 
         [0060]    B 2 : Cell separation centrifugation at a first speed is then performed as described above until the red blood cells  24  are consolidated at the bottom of compartment  12  and the MNC band  25  stays on top of the filter  9  at the interface between plasma  26  and density gradient medium  13 . 
         [0061]    B 3 : The stopper  29  is then removed and replaced by a flexible container  23 . 
         [0062]    B 4 : Continued centrifugation at a second speed displaces the plasma  26  into the container  23 , the syringe cylinder  2  being forced downwards to contact the bottom  8   a  of the supporting bucket  8 . Optionally, this displacement of the plasma may instead be done manually or automatically in a dedicated holder or apparatus similar to, or the same as that outlined with reference to subfigure A 10  in  FIG. 2A . Harvesting of the MNC band is then carried out as described with reference to subfigures A 9  to A 12  above. 
       Second Embodiment 
     Step Density Gradient Separation 
       [0063]    Another embodiment of the syringe device of the present invention is illustrated in  FIGS. 3 to 6 . With specific reference to  FIG. 3 , the syringe device, generally designated by the reference numeral  31 , similarly to the syringe device in  FIGS. 1 and 2A ,  2 B comprises a syringe cylinder  32  in which a plunger or piston including a piston plug  33  and a piston rod  34  is slidingly mounted. The top end  35  of the cylinder  32  tapers to a sample inlet/outlet  36 , whereas the bottom end  37  of the cylinder is open. In the illustrated case, the inlet/outlet  36  is capped by a stopper  38 . A partition member  39  in the form of a filter or grid, for example, in the following for simplicity referred to as filter  39 , is attached to the piston plug  33  and divides the volume enclosed between the piston plug  33  and the inlet/outlet  36  into a sample compartment  41  above the filter  39  and a compartment  42  below the filter  39  for containing a density gradient medium  43 . 
         [0064]    In contrast to the embodiment in  FIGS. 1 and 2A ,  2 B, the filter  39  in the syringe device  31  is mounted to the piston plug  33  such that the distance between the filter and the piston plug is adjustable to enable the filter  39  to be displaced relative to the piston plug  33  from an outermost position where the filter is at a fixed distance from the piston plug, to an innermost position where the filter is considerably closer to the piston plug. The mounting of the filter to the piston in order to permit such displacement or “collapse” of the filter  39  may be designed in various ways and may be made to take place in a continuous or stepwise manner, such as in a single step. The means to cause such collapse of the filter may also be designed in various ways depending on the mounting of the filter to the piston plug. 
         [0065]      FIG. 4  schematically shows an embodiment of a filter-piston plug mounting where collapse of the filter may be caused when a force exceeding a predetermined value acts on the filter, such as e.g. the force on the filter when the syringe device  31  is centrifuged at a sufficiently high speed. In  FIG. 4 , the filter  39  is attached to the piston plug (not shown) by a cylindrical two-part member  40  consisting of a cylindrical filter holder  44  and a cylindrical support  45  fixed to the piston plug (not shown). The holder  44  has a slightly larger internal diameter than the external diameter of the support  45 , so that collapse of the filter may be caused by overcoming the friction between the holder  44  and the support  45 . In the collapsed position, the filter  39  is adjacent to or rests on the top edge of support  45 . 
         [0066]    In a variant (not shown), the holder  44  is threadedly engaged with the support  45  so that collapse of the filter may be accomplished by rotating the piston plug (via the piston rod) relative to the filter  39 . Numerous other variants are conceivable to the skilled person. 
         [0067]    A method embodiment of using the syringe device illustrated in  FIGS. 3 and 4  in a step density gradient separation process will now be described with reference to  FIG. 5 , where the different method steps are schematically illustrated by subfigures A 1  to A 8 . 
         [0068]    A 1 : The syringe is placed in a dedicated holder (not shown), and a tube  46  connected to a container  47  with sample  48  is attached to the syringe inlet/outlet  36 . The holder is provided with actuation means (not shown) for actuating the piston rod  34  to displace the piston plug  33  vertically within the syringe cylinder  32 . Such means may be manual or automated. The actuation means is then operated to initiate displacement of the piston plug downwards as indicated by arrow  49 . As shown in subfigure A 2  below, the filter  39  prevents mixing of the sample with the density gradient medium  43 . 
         [0069]    A 2 : All sample  48  has now been sucked into the expanded compartment  41  of the syringe, the filter  39  having moved together with the piston plug  33  with the density gradient medium  43  enclosed between them. In the illustrated case, the end of the piston rod  34  has reached the level of the open end of the syringe cylinder  32 . 
         [0070]    A 3 : The syringe device is then capped by applying a stopper  50  to the syringe inlet/outlet  36 , removed from the holder device, and put in a centrifuge (not shown) and centrifuged. During the centrifugation, the filter  39  “collapses”, i.e. is displaced towards the piston plug  33  by the centrifugal force making the filter holder  44  ( FIG. 4 ) overcome the friction and slide down the support  45  ( FIG. 4 ), simultaneously as the sample components are separated. 
         [0071]    A 4 : When the centrifugation is completed, (in the case of the sample being blood) the red blood cells are consolidated in a layer  51  on top of the piston plug  33 , whereas the plasma  52  is above density gradient medium  43 . The MNH cells are banded in layer  53  at the plasma/density gradient medium interface. The filter  39  is in its collapsed position close to the piston plug  33 , so that the MNC cell band  53  is at a considerable distance from the filter  39 . 
         [0072]    A 5 : The syringe device is then again put in the dedicated holder (not shown) referred to above in connection with step A 1 , so that the piston rod  34  can be actuated to move upwards as indicated by arrow  54 . The stopper  50  is removed, and a tubing  55  is connected to the inlet/outlet  36  of the syringe device. Optionally, an optical detector  56  is mounted on the tubing  55 . A number of containers for collection of fluid expelled from syringe device, here three collector tubes  57   a - 57   c , are provided at the other end of the tubing  55 . Fractionation of the syringe contents is initiated by actuation of the piston rod  34  in the holder with the tubing  55  opening into collector tube  57   a.    
         [0073]    A 6 : The piston plug  33  is moved upwards, while the first fraction, i.e. the cell-free plasma  52 , is collected in the first collector tube  57   a . In the Figure, the MNC band  53  has almost reached the top end of the syringe cylinder  32 . 
         [0074]    A 7 : Further displacement of the piston plug  33  upwards in the syringe cylinder  32  expels the MNC band  53  out of the syringe device into the tubing  55 . This is detected by the detector  56  (or optionally visually) and the end of tubing  55  is moved to open into the second collector tube  57   b  for harvest of the MNC cells  53  therein. This may be done manually, but may also be done automatically by the holder device being triggered by the detector  56 . 
         [0075]    A 8 : When the MNC cell band  53  has been completely expelled from the syringe device as detected by detector  56  (or visually) and collected in collector tube  57   b , the end of tubing  55  is moved to the third collector tube  57   c  for collection of waste density gradient medium  43  which is expelled on continued upwards displacement of the piston plug  33  until the filter  39  is at its top position in syringe cylinder  32 . 
       Linear Gradient Separation 
       [0076]    Use of a syringe device similar to that shown in  FIG. 3  in a linear density gradient separation process will now be described with reference to  FIG. 6 , where the different method steps are schematically illustrated by subfigures A 1  to A 11  (identical reference numerals being used for corresponding parts). In the subfigures, the syringe device, generally designated by the reference numeral  61 , is placed in the dedicated holder mentioned above or in a corresponding device (not shown). 
         [0077]    In the different subfigures, the syringe device  61 , similarly to the syringe device in  FIGS. 1 to 3 , comprises a syringe cylinder  62  in which a plunger or piston including a piston plug  63  and a piston rod  64  is slidingly mounted. The top end of the cylinder  62  tapers towards a sample inlet/outlet  66 , whereas the bottom end of the cylinder is open. A partition member  69  in the form of a filter or grid, for example, in the following for simplicity referred to as filter  69 , is attached to the piston plug  63  and divides the volume enclosed between the piston plug  63  and the inlet/outlet  66  into a sample compartment  71  above the filter  69  and a compartment  72  below the filter  69  for containing a density gradient medium. 
         [0078]    While the filter mounting shown in  FIG. 4  could per se also be used here, it is preferable to use a mounting structure capable of collapsing to a higher degree, such as may, e.g. be obtained by a telescopic assembly having more than two cylinders that slidingly fit into each other. 
         [0079]    A 1 : With the syringe device  61  placed in the dedicated holder, the syringe inlet/outlet  66  is then connected via a tube  70  to a gradient mixer  74  comprising a first container  75  with a high density medium (HDM), which e.g. may be a PERCOLL™ or FICOLL™ medium with high density, and a second container  76  with a low density medium (LDH), which e.g. may be a PERCOLL™ or FICOLL™ medium with low density. Fluid from the gradient mixer  74  is introduced into the syringe device by actuating the piston rod  64  for downward movement of the piston plug  63  as indicated by arrow  77 . When the piston plug  63  is moved downwards, the filter  69  in the collapsible assembly remains at the top of the syringe cylinder  62 . 
         [0080]    A 2 : The gradient mixer  74  has been emptied and a linear gradient of density medium  73  has been formed in the compartment  72  between the filter  69  and the piston plug  63 . 
         [0081]    A 3 : A tube  78  connected to a container  79  with sample  80  is then attached to the syringe inlet/outlet  66 , and the piston rod  64  is again actuated for downward movement of the piston plug  63 , as indicated by arrow  77 . Optionally, the sample has first been subjected to a pre-incubation with beads containing immobilized specific affinity ligands for depletion of unwanted cells. 
         [0082]    A 4 : All sample  80  has now been sucked into the expanded compartment  71  of the syringe, the filter  69  having moved downwards together with the piston plug  63 . 
         [0083]    A 5 : The syringe device is then capped with a stopper  81 , removed from the holder device, put in a centrifuge (not shown) and centrifugation is started. During centrifugation, the filter  69  collapses (i.e. is displaced towards the piston plug  63 ) and the sample components are separated. When the centrifugation is completed, the filter  69  is at its bottom position, and (in case the sample is blood) the red blood cells are consolidated in a layer  82  on top of the piston plug  63 . The MNC cells are fractionated into several bands, here three cell bands  83   a - 83   c , below the plasma  84 , all within the linear density gradient medium  73 . 
         [0084]    A 6 : The syringe device is then again put in the dedicated holder (not shown) referred to above, so that the piston rod  64  can be actuated to move upwards as indicated by arrow  85 . The stopper  81  is removed, and a tubing  86  is connected to the inlet/outlet  66  of the syringe device. Optionally, an optical detector  87  is mounted on the tubing  86 . A number of containers for collection of fluid expelled from the syringe device, here five collector tubes  88   a - 88   e , are provided at the other end of the tubing  86 . Fractionation of the syringe contents is initiated by actuation of the piston rod  64  in the holder device with the tubing  86  opening into collector tube  88   a.    
         [0085]    A 7 : The piston plug  63  is moved upwards, while the first fraction, i.e. the cell-free plasma  84 , is collected in the first collector tube  88   a  together with a top portion of the density gradient medium  73 . 
         [0086]    A 8 : Further displacement of the piston plug  63  upwards in the syringe cylinder  62  expels the first cell band  83   a  out of the syringe device into the tubing  86 . This is detected by the detector  87  (or optionally visually) and the end of tubing  86  is moved to open into the second collector tube  88   b  for harvest of the cell band  83   a  therein. This may be done manually, but may also be done automatically by the holder device being triggered by the detector  87 . 
         [0087]    A 9 : Similarly as above, the second cell band  83   b  is expelled from the syringe device, detected and collected in collector tube  88   c  by further displacement of the piston plug  63  upwards in the syringe cylinder  62 . 
         [0088]    A 10 : Similarly as above, the third cell band  83   c  is expelled from the syringe device, detected and collected in collector tube  88   d  by further displacement of the piston plug  63  upwards in the syringe cylinder  62 . 
         [0089]    A 11 : Finally, when the third cell band  83   c  has been expelled from the syringe device and collected in collector tube  88   d , the end of tubing  86  is moved to the fifth collector tube  88   e  for collection of waste density gradient medium  73  expelled on continued upwards displacement of the piston plug  63  until the filter  69  is at its top position in syringe cylinder  62 . 
         [0090]    It is to be understood that the invention is not limited to the particular embodiments of the invention described above, but the scope of the invention will be established by the appended claims.