Abstract:
The invention relates to a method and a device for separating blood using a centrifuge in order to obtain different blood fractions. A separation container may be filled with blood via a feed connection that can be aseptically connected to the container, the relevant blood fraction may be removed from the separation container via a removal connection that can be aseptically connected to the container and both the feed device and the removal device folln a respective closed system during the sterile connection to the separation container. The separation container may have at least one feed connection that can be aseptically connected to the container and at least one removal connection that can be aseptically connected to the container, to which hermetically sealed feed and removal devices can be aseptically attached in a sterile manner.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a divisional of U.S. application Ser. No. 13/062,899, filed May 26, 2011, and entitled “Method and Device for Separating Blood Using a Centrifuge,” which application is a national stage application filed under 35 U.S.C. §371 of International Application No. PCT/DE2009/001273 filed Sep. 13, 2009, which application claims priority from German Patent Application No. 10 2008 047 068.6, filed Sep. 12, 2008, each of which is incorporated by reference herein in its entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The invention relates to a method for separating blood using a centrifuge in order to obtain different blood fractions. 
         [0004]    Furthermore, the invention relates to a device for separating blood using a centrifuge comprising a rotor and at least one separation container, in particular to carry out the method. 
         [0005]    2. Description of Related Art 
         [0006]    Blood can be separated into different blood fractions by means of a centrifuge in that the centrifuge is rapidly rotated at defined revolutions and over a defined period of time. After the centrifugation, the blood in the separation container has changed. The individual constituents have separated into blood plasma, which consists primarily of protein and water, white blood corpuscles and solid blood constituents. 
         [0007]    The preparation of the blood before and after the separation process is very time consuming and is generally done in the atmosphere—that is, in an open system that is, by definition, contaminated. It is precisely in the case of umbilical cord blood preparation, but also in the case of other kinds of blood, that the contamination is not desired. For more comprehensive preparations, the white blood corpuscles are the most relevant. 
         [0008]    Working on the basis of the prior art known from practical application, the object of the invention is to provide a method and a device of the type under discussion that eliminates the risk of contamination during the filling with blood and during the extraction of blood fractions. 
       SUMMARY OF VARIOUS EMBODIMENTS 
       [0009]    The aforesaid object is achieved with a method exhibiting the features of various embodiments described herein. Correspondingly a method of the type under discussion is configured in such a way that the separation container is filled with blood via a feed port, which can be aseptically connected to said separation container; that the relevant blood fraction is extracted from the separation container via an extraction port, which can be aseptically connected to said separation container; and that the feed device and the extraction device form a respective closed system during the sterile connection to the separation container. 
         [0010]    The aforementioned object is achieved with a device exhibiting the features of various embodiments described herein. Correspondingly a device of the type under discussion is configured in such a way that the separation container has at least one feed port, which can be aseptically connected to said separation container, and at least one extraction port, which can be aseptically connected to said separation container, to which the hermetically sealed feed and extraction devices can be aseptically attached. 
         [0011]    It has been recognized from the prior art known from practical experience that the risk of contamination can be greatly reduced if the filling of the separation container, which is outwardly sealed in the well-known manner and which is located in the centrifuge, and the extraction from this separation container of the centrifuge are performed with the exclusion of the atmosphere. The invention recognizes that it is possible to exclude contact with the atmosphere, if a closed system is formed between the feed device and the separation container and between the extraction device and the separation container, and/or if the aseptically connectable ports provide such a condition that the specifically designed and hermetically sealed feed and extraction devices can be aseptically attached. It is apparent that the inventive approach reduces the risk of contamination. 
         [0012]    According to a preferred embodiment two feed devices could be connected to two feed ports for feed purposes. This strategy offers the possibility of also delivering, in addition to blood, auxiliary agents to improve the centrifugation. Hence, it would also be possible to aseptically weld a feed device for the anticoagulated blood and a feed device for the hydroxyethyl starch solution (hereinafter referred to as HES) to the respective feed port. The HES promotes the sedimentation of erythrocytes or rather red blood corpuscles that are to settle out. 
         [0013]    While the separation container is being filled with blood, it would be advantageous to simultaneously attach the feed device for the HES. This strategy has the advantage that the sterile air, which is displaced from the separation container by the blood, can escape into the feed device for the HES. Moreover, while the blood is injected by means of the feed device for the HES, a negative pressure could also be generated, and the sterile air could be actively drawn in from the separation container. The displaced or aspirated sterile air could flow into a supply container of the feed device for the HES or could be released into a pressure-compensating device. This approach ensures that the sterile air flows into the supply container of the feed device for the HES in such a way that it bypasses the HES. To this end, during the delivery of sterile air, the HES-containing area of the flexible supply container of the feed device for HES could be moved away from the inlet point to the supply container. For example, the supply container, which exists as a flexible bag, could be bent away, so that the displaced sterile air flows in without flowing through the HES. In the case of the HES addition, sterile air displaced by the HES solution could flow just as well into the feed device for blood. 
         [0014]    This particular design of the feed device could enable a volume-accurate addition of the blood. The same applies to the addition of the HES solution. The necessary amount of HES could be pre-calculated in consideration of the amount of injected blood. 
         [0015]    When the filling with blood and in some cases additionally with HES has been completed, the sterile disconnection between the feed device and the separation device could occur. This step could be achieved by the well-known sterile welding method. In any event it is absolutely imperative that the separation container be sealed off from the outside world. 
         [0016]    The separation container has to absorb many forces during centrifugation and for this reason has a shell structure for stabilization. The shell structure could comprise a cover plate that exhibits recesses for accommodating the feed ports and optionally the extraction port. After the disconnection, the freely downward-extending ports can be brought into the recesses, so that there are no breakdowns during the centrifugation. Centrifugation occurs in exact time phases, in particular at an acceleration of 1,500 g. 
         [0017]    The separation container could hold two containers that are flow-connected to each other. During the centrifugation, the blood fraction of white blood corpuscles collects predominantly inside the flow connection between the containers. This state can be detected by the color of the substance. In addition, an optical reader could be used to determine the phase boundary. When the phase boundary is recognized, the flow connection that is present as a plastic hose could be disconnected by means of a hose clamp at the phase boundary between the blood fraction of the solid blood constituents and the blood fraction of white blood corpuscles. The hose clamp could be applied by means of the open regions of the shell structure. The separation container itself is sealed air tight and can be connected to the systems, which are also closed, only by way of the ports. The design of the flow connection per plastic hose is advantageous because of the ease with which it can be disconnected. At the same time the hose clamp can be positioned exactly at the point, where the phase boundary runs between the erythrocytes and the leukocytes. This site may vary from patient to patient. Experience has demonstrated that in forming the separation container with the plastic hose the yield is very high. In addition, the extraction of blood fractions does not have to be limited to the leukocytes. Rather plasma may also be aspirated from the top internal container in order to reach a standardized volume. The standardized volume can be used for cryopreservation, for which there are narrow specifications for the procedure. 
         [0018]    According to a second embodiment, there could also be an additional feed port for an additional feed device—in this case, for delivering a filling material to reduce the volume of the container that holds the centrifuged erythrocytes. In order to form the phase boundary inside the flow connection, preferably at the lower end of the flow connection, between the containers, the volume of the lower container could be changed. The change relates to the fact that there is a difference between the fill volume of blood and the erythrocytes therein and the pack volume of the centrifuged erythrocytes. The future pack volume can be calculated by means of the hematocrit value, known from each blood sample, and by means of the fill volume of the blood. The fill volume can be determined either by using the value of the known volume of the feed device (or more specifically the bag volume) or by using a scaled feed device, which results in the amount of drawn blood that then yields the fill volume. As an alternative, the separation container can also be scaled. The volurfte of the container of the separation container, which is provided for holding the centrifuged erythrocytes, could be decreased by filling with inert beads. After deteanining the fill volume in the separation container and after calculating the pack volume by means of the hematocrit value, the percentage, to which the lower container will be filled with the centrifuged erythrocytes, can be estimated. The expected pack volume of erythrocytes usually falls below the volume of the lower container of the separation container. In order to form at this point the phase boundary in the flow connection and not in the lower container, the container could be filled with so many beads that the volume is filled beyond the expected pack volume. During centrifugation the beads collect on the bottom of the container under discussion due to their comparatively high weight, and over them the erythrocytes. The phase boundary develops, as desired, in the flow connection, at its lower end. Therefore, the leukocytes or more specifically the so-called buffy coat, consisting of leukocytes and thromocytes, collect optimally in the flow connection. 
         [0019]    In order to extract the blood fraction of white blood corpuscles from the plastic hose, an extraction device could be aseptically connected to the extraction port. The extraction device could exhibit a configuration described in DE 10 2008 035 835, wherein the interior of the extraction device has an internal line. After the two closed systems have been connected to form a total closed system, this inner line could be inserted into the separation container or more specifically into the flow connection, in order to extract the blood fraction of white blood corpuscles. The interior of the extraction device could have a syringe that extracts the relevant blood fraction with one plunger stroke and then transfers it into the hermetically sealed extraction device. The interior of the extraction device could have, for example, a container to hold the blood fraction. 
         [0020]    After the blood fraction of white blood corpuscles has been removed from the separation container, the extraction device could also be aseptically disconnected, so that it is ensured that in any event the extraction device does not make contact with the atmosphere. 
         [0021]    With respect to the construction of the device, the first step is to stress the special configuration of the separation container according to a preferred embodiment. In the interior of the separation container the separation container could comprise two containers that are flow-connected to each other. When the separation container is in quiescent position in the rotor bowl of the rotor, the first container with a funnel-shaped bottom could be disposed above the second container. The flow connection could be attached to the funnel outlet in the form of a plastic hose that empties into the second container. The funnel-shaped bottom facilitates the delivery of blood from the first to the second container. As an alternative to the above-described configuration, there could also be provided a ring-shaped separation container that extends about the rotor axis and has a cross section that consists of two expansions and a central constriction. Furthermore, the ring-shaped separation container could also be segmented into a plurality of chambers. 
         [0022]    Since the blood fraction of white blood corpuscles is relevant for the subsequent preparation steps, and these white blood corpuscles are collected predominantly in the flow connection after the centrifugation, the extraction port could be in alignment with the flow connection. This feature makes it much easier to insert an inner line of the extraction device after the sterile connecting to the extraction port. 
         [0023]    The stability of the separation container is not especially high because of the soft plastic hose as the flow connection. Therefore, the separation container could be disposed inside a shell structure. The shell structure could have open areas, through which a hose clamp can be fastened on the flow connection or more specifically the plastic hose. The hose clamp is supposed to prevent the phases that were obtained after the centrifugation—that is, the red blood corpuscles in the lower internal container and the white blood corpuscles in the plastic hose or more specifically in the flow connection—from mixing. 
         [0024]    As stated above with respect to the method, it would be possible to provide, according to a first embodiment, two feed ports, which are spaced at a distance from the extraction port and are intended for blood, on the one hand, and for the hydroxyethyl starch solution HES, on the other hand. In the specific case it would be possible to use a feed device that comprises a syringe, sitting inside a sleeve and comprising a tube and a plunger that exhibits a passage opening. If there are additionally shut-off devices at the tube and the plunger end, then it is possible to draw in and eject multiple times, during which the shut-off devices are either alternatively closed or opened, as needed. Consequently it is possible to work in both directions, because the suction is not necessarily a function of pulling the plunger out of the tube. Owing to the passage opening in the plunger the suction process can also take place by pushing the plunger in; only the correct shut-off device has to be opened—that is, the shut-off device on the suction side. Furthermore, it is important that aseptically connectable ports descend downwards from the sleeve. Additional possibilities for closed donor systems with downwards descending and aseptically connectable ports are apparent from DE 10 2008 035 837. 
         [0025]    According to a second embodiment, the separation container could have an additional aseptically connectable feed port, to which an additional hermetically sealed feed device for a filling material, in particular inert beads, can be attached in order to reduce the volume in the container that is provided for holding the centrifuged erythrocytes. The feed devices could be operated subject to gravitational force and could have in each case a metering shut-off device so that a volume-accurate metering can be performed. 
         [0026]    According to an additional embodiment for an extraction device with the above-described inner line, which can be applied in the flow connection, this inner line could be connected to a syringe that is situated inside a sleeve and comprises a tube and a plunger, which has a passage opening. The inner line can be handled via a bellows of the sleeve; for this purpose the aseptically connected ports form the guide for the inner line. Then the syringe is handled by way of the shut-off devices, as described with respect to the feed device. 
         [0027]    One important aspect of the invention is that the feed device and the extraction device are in themselves closed systems that also faun together with the separation container a closed system during the aseptic connection to the corresponding port of the separation container. As a result, contamination is no longer a factor 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0028]    At this point there are a plethora of possibilities for configuring and further developing the teaching of the present invention in an advantageous way. To this end, reference is made, first of all, to the various embodiments described herein and secondly to the following description of two embodiments of the invention with reference to the drawings. The description of the inventive embodiments presented herein also includes an explanation of the generally preferred configurations and further developments of the teaching. In the drawing 
           [0029]      FIG. 1  is a diagrammatic sketch of a top view of the centrifuge in its operating position using the inventive device with a separation container. 
           [0030]      FIG. 2  is a schematic drawing, enlarged, of a detail from  FIG. 1 , regarding a first exemplary embodiment of the inventive device with two feed ports and one extraction port. 
           [0031]      FIG. 3  is a schematic drawing, reduced in size, of the subject matter from  FIG. 2  with two aseptically connected feed devices. 
           [0032]      FIG. 4  is a sketch of an alternative removal of the displaced sterile air from the separation container. 
           [0033]      FIG. 5  is a schematic drawing, reduced in size, of the subject matter from  FIG. 2  prior to the aseptic connection to an extraction device. 
           [0034]      FIG. 6  is a schematic drawing, reduced in size, of the subject matter from  FIG. 2  with the aseptically connected extraction device and the hose clamp as well as the inserted inner line. 
           [0035]      FIG. 7  is a schematic drawing of the feed devices at the separation container, according to a second exemplary embodiment of the device according to the invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0036]      FIGS. 1 to 3, 5 and 6  show a device for separating blood using a centrifuge (shown in  FIG. 1 ) with a rotor  2  and herein four rotor bowls  3 . Each rotor bowl  3  has a separation container  4 . The drawing does not show the rotor shaft or other design features. 
         [0037]    The first exemplary embodiment of the invention provides that the separation container  4  has two feed ports  5 ,  6 , which can be aseptically connected to the separation container, and an extraction port  7 , which can be aseptically connected to the separation container, to which the hermetically sealed feed and extraction devices  8 ,  9 ,  10  can be aseptically attached. 
         [0038]    The separation container  4  comprises two containers  11 ,  12 , which are flow-connected to each other. The bottom of the container  11  is configured as a funnel. In order to form the flow connection  13 , there is a flexible PVC hose that is seal-welded to the containers  11 ,  12 . The extraction port  7  aligns with the flow connection  13 . The separation container  4  is disposed inside the shell structure  14  comprising a base plate  15  and a cover plate  16 . The base plate  15  and the cover plate  16  are securely connected to the shell structure. The shell structure  14  has reinforcements  17  that fulfill the function of inner shoulders or stabilizers. 
         [0039]    The shell structure  14  has openings (not illustrated here), through which the hose clamp  18  (shown in  FIG. 6 ) can be fastened on the flow connection  13 . 
         [0040]      FIG. 3  shows that the umbilical cord blood with an anticoagulant  19  flows through the feed device  8  into the separation container  4 , and through the feed device  9  a hydroxyethyl starch solution  20  flows, hereinafter referred to as HES. 
         [0041]    The feed devices  8 ,  9  have a syringe  22 , which sits inside a sleeve  21  and comprises a tube  23  and a plunger  24 , which has a passage opening  25 . The reference numeral  35  denotes a pressure-compensating device, into which the displaced sterile air can escape. 
         [0042]    From the sleeve  21  aseptically connectable ports  27  descend, which can be opened and closed by shut-off devices  28 ,  29  in the form of two way stopcocks. The shut-off devices  28 ,  29  serve to achieve the transfer of the umbilical cord blood with anticoagulant  19  from the supply container  30  to the separation container  4  and to achieve the transfer of the HES  20  from the supply container  31  to the separation container  4 . 
         [0043]    The respective substance  19 ,  20  is drawn into the respective feed device  8 ,  9  through the syringe  22 . The respective substance  19 ,  20  can be transferred into the separation container  4  through the passage opening  25 . The tube-sided port  32  and the plunger-sided port  27  extend downwards from the sleeve  21  and are tightly connected to it. 
         [0044]    The operating principle of the feed devices  8 ,  9  is explained below. 
         [0045]    In the present embodiment the supply containers  30 ,  31  and the syringe  22  with the sleeve  21  are pre-assembled. The plunger-sided port  27  is attached to the feed port  5 ,  6  of the separation container  4 . 
         [0046]    The shut-off device  29  of the tube-sided port  32  is opened prior to the—in this case, volume-accurate—suction process, and the shut-off device  28  of the plunger-sided port  27  of the syringe  22  is closed. The syringe  22  or more specifically the plunger  24  and the shut-off devices  28 ,  29  are actuated by means of the sleeve  21  from the outside. 
         [0047]    During the suction process the tube  23  of the syringe  22  is filled with the substance  19 ,  20 . 
         [0048]    Prior to passing the substance  19 ,  20  from the syringe  22  to the separation container  4 , the shut-off device  29  of the tube-sided port  32  is closed, and the shut-off device  28  of the plunger-sided port  27  is opened. In order to pass the substance  19 ,  20  even further, the plunger  24  of the syringe  22  is pushed into the tube  23 . As a result, the tube  23  empties via the passage opening  25  and the plunger-sided port  27  and its opened shut-off device  28  into the feed port  5 ,  6  and then into the separation container  4 . Reference numeral  33  denotes the sterile connection between the feed ports  5 ,  6  and the plunger-sided ports  27 . 
         [0049]      FIG. 4  shows, as an alternative to the shut-off device  29 ,  28  in the form of a two way stopcock, a three-way stopcock  34  with a pressure-compensating device  35  for accommodating the displaced sterile air from the separation container  4 . The feed devices  8 ,  9  and the extraction device  10  also have a pressure-compensating device  35  for buffering the variations in volume. 
         [0050]      FIGS. 5 and 6  relate to the extraction process after the centrifugation. The feed devices  8 ,  9  are already aseptically welded; the feed ports  5 ,  6  have become a little shorter. The extraction device  10  has an inner line  36  that can be applied through the extraction port  7  into the flow connection  13 . The inner line  36  is disposed inside a bellows  37  with a pressure-compensating device  35  and is aseptically connected to the sleeve  21  with a syringe  22  comprising a tube  23  and a plunger  24  that has a passage opening  25 . The syringe  22  of the extraction device  10  works according to the same principle as the syringe of the feed devices  8 ,  9 . In this case, too, the shut-off devices  28 ,  29  are provided. The only difference is that the tube  23  points toward the separation container  4 , where, in addition to the corresponding actuation of the shut-off devices  28 ,  29 , the suction process takes place before the extracted blood fraction of white blood corpuscles is emptied via the passage opening  25  of the plunger  24  into the collapsed bag  38 . The bag  38  is also aseptically connected to the plunger-sided port  27  by way of the port  40  of the bag. 
         [0051]    In  FIG. 5  the inner line  36  is still wound up. In  FIG. 6  the inner line  36  is extracted and inserted into the flow connection  13 , in which the relevant white blood corpuscles have collected. In addition,  FIG. 6  also shows a stabilizing sleeve  39  at the extraction port  7 . 
         [0052]    The feed devices  8 ,  9  and the extraction device  10  are closed systems, just like the separation container  4 . During sterile connection to the corresponding ports  5 ,  6 ,  7  of the separation container  4 , the feed devices  8 ,  9  and the extraction device  10  also form together with said separation container a closed system. 
         [0053]    The following process steps can be performed with this device. 
         [0054]    Umbilical cord blood with an anticoagulant  19  and the HES  20  is fed into the separation container  4  by way of the two feed devices  8 ,  9 . At the same time sterile air from the separation container  4  flows into the feed device  9  for HES  20  and, in particular, as far as into the supply container  31 . In  FIG. 4  a three-way stopcock  34  with a pressure-compensating device  35  is provided as an alternative. The displaced sterile air flows into the supply container  31  of the feed device  9  for HES  20 , so that it flows past the HES  20 . During the delivery of the sterile air, the HES-containing area of the flexible supply container  31  of the feed device  9  for HES  20  is moved away from the inlet point  42  into the supply container  31 . The next step involves a volume-accurate addition of both substances  19 ,  20 . The required amount of HES  20  is calculated in consideration of the added amount of umbilical cord blood with an anticoagulant  19 . Having filled the separation container  4 , the feed devices  8 ,  9  are aseptically disconnected. In order to extract the blood fraction of white blood corpuscles from the flow connection  13 , the extraction device  10  is aseptically connected to the extraction port  7 . The inner line  36 , disposed in the interior of the extraction device  10 , is inserted into the flow connection  13 , in order to extract the blood fraction of white blood corpuscles. After the blood fraction of white blood corpuscles has been extracted through repeated aspiration and ejection from the separation container, the extraction port  7  and the port  42  of the bellows  37  of the extraction device  10  are aseptically disconnected. 
         [0055]    The second embodiment (shown in  FIG. 7 ) of the device according to the invention relates to a third aseptically connectable feed port  43 , to which an additional hermetically sealed feed device  44  for feeding inert beads  45  is attached. The inert beads  45  are needed to reduce the volume in the container  12 , so that the phase boundary between the erythrocytes, which are supposed to collect predominantly in the container  12  after the centrifugation, and the leukocytes, which are supposed to collect exclusively in the flow connection  13  after the centrifugation, acquires a predefined position on the lower end of the flow connection  13 . 
         [0056]    All three feed devices  8 ,  9 ,  44  are operated herein subject to gravitational force. A metering shut-off device  46  is provided for metering the blood  19 , HES  20  and inert beads  45 . 
         [0057]    With respect to features that are not shown in the figures, reference is made to the general part of the specification. 
         [0058]    Finally it must be pointed out that the teaching according to the invention is not limited to the above-described embodiment.