MXPA97004014A - Centrifuga with anu filter - Google Patents

Abstract

The present invention relates to a device for separating components such as fibrin monomer from blood by centrifugation around a central axis, comprising a first annular chamber (70) defined by an outer cylindrical wall (72) and a cylindrical wall interior (71) both walls that are arranged concentrically around an axis of rotation, and by a wall (73) of the upper part and a wall (74) of the bottom, where the wall of the phono is formed by a piston (55) displaceable within the first. The device further comprises a second chamber (75) below the first chamber and having communication with the first chamber through a first conduit (4) and defined by another cylindrical wall (72), the bottom wall. (74) of the first chamber and another wall (76) of the bottom. The device also comprises a capsule (45) accommodated in the second chamber and including a plurality of compartments to receive the respective positions that promote separation.

Description

CENTRIFUGA WITH ANULAR FILTER TECHNICAL FIELD The invention relates to a centrifuge device for separating a component, such as a fibrin monomer from plasma, the method comprising treating the plasma with one or more reagents, wherein the reagents are distributed to a suitable reaction chamber containing the plasma and where these reagents are therefore removed from a desired product by means of the new device and centrifugal filtration method.

PREVIOUS TECHNIQUE EP-PS No. 592,242 discloses methods and compositions for a completely new fibrin sealant comprising contacting a desired site with a composition comprising the fibrin monomer and converting this monomer to a fibrin polymer concurrently with the contact step . The term "fibrin" is defined as fibrin I, fibrin II, and / or des-jff? '- fibrin. REF: 24856 Additionally, a method is known from US Patent Application Serial No. 155,984 for separating a component, such as fibrin monomer from blood. This method for separating the components of a liquid containing various components of variable specific weights comprises the steps of the blood that is collected in a first chamber of a device, this chamber being defined by an axially symmetrical external and internal wall, so substantial The blood is subjected to a centrifugation by means of the rotation of the device around the axis of symmetry of the chamber to establish a concentric interface between the components of the blood. At least one of the components of the blood such as the plasma is subsequently transferred to a second chamber in the device preferably by means of reducing the volume of the first chamber during a continuous centrifugation of the device. The axially substantially symmetrical inner wall is provided in the first chamber to ensure that all blood is subjected to a centrifugal rotation necessary for separation. This inner wall is of a radius adapted to the desired speed of rotation.

In the second chamber, a fraction with non-crosslinked fibrin polymer is separated from the plasma by means of a suitable enzyme and subsequently redissolved in the fibrin monomer and transferred to a syringe through a filter by reducing the volume of the second chamber. However, the output in the separation of a component, such as fibrin I from the blood, only by means of filtration in a device of the above type does not provide a satisfactory result. This is mainly due to the fact that it is difficult to ensure a satisfactory separation of the fraction containing fibrin I in the second chamber, and consequently, a relatively high amount of the blood content of fibrin I is lost during the second transfer of a fibrin. fraction of fluid from the second chamber to the first chamber during the next step of the method. Also, in the above method of fibrin monomer, the above-described treatment of fibrinogen within the plasma with a suitable enzyme produced the non-crosslinked fibrin polymer in the form of a thick gel mass at the bottom of the second chamber. To provide the desired solution of fibrin monomer, a significant amount of redissolution buffer combined with substantial agitation was required. This resulted in several disadvantages. First, preferred methods of fibrin monomer, for example, for use as a fibrin sealant as in EP 592,242, required concentrated solutions of fibrin monomer, and the large amount of redissolution buffer or solvent required to dissolve the mass of the fibrin monomer. gel provided diluted solutions that did not work well. In addition, the substantial agitation required to dissolve the gel mass in the fibrin monomer solution can cause damage to the device and fibrin itself. A co-pending application entitled "Method and Device for Separating a Such Fibrin I From Blood Disease" filed concurrently with the present invention describes an invention that includes a method comprising separating the non-crosslinked fibrin polymer from a plasma fraction in a cylindrical chamber, carried out during the centrifugation whereby the non-crosslinked fibrin polymer is deposited on the outer wall of the chamber. Subsequently the fraction of remaining fluid collected in the chamber is removed from the chamber, and the fraction with the non-crosslinked fibrin polymer remaining in the chamber deposited substantially in the wall is caused to dissolve by the addition of a solvent and by centrifugal agitation. Since treatment of the plasma with the enzyme is carried out during continuous centrifugation, the centrifugal force on the resulting non-crosslinked fibrin polymer provides that it is precipitated as a thin gel film which sticks substantially to the circumferential walls of the camera.' The remaining plasma liquid is deposited at the bottom of the chamber when it separates the centrifugation and can be removed by any convenient means. The desired solution of fibrin monomer is subsequently provided by introducing a suitable buffer solution of redissolution in the chamber and by subjecting the buffer in the gel-coated chamber to centrifugal agitation. This method provides advantage over the previous methods. First, redissolution of the non-crosslinked gel by the buffer is extremely efficient due in part to the large surface area of the same volume of the fibrin gel compared to the fibrin gel mass provided in the above methods. Accordingly, the gel can be dissolved with small amounts of redissolution buffer resulting in a properly concentrated fibrin monomer solution. In addition, the action of centrifugal agitation in the buffer solution within the gel-coated chamber is a completely benign method that does not cause damage to the equipment or the fibrin monomer product. The copending invention also includes a method comprising feeding blood preferably in the presence of an anticoagulant to a first annular chamber in a device, wherein the annular chamber is defined by a cylindrical outer wall and a cylindrical inner wall, both walls being they extend coaxially around a common axis, as well as by a wall of the upper part and a bottom wall, where the wall of the upper part or the bottom wall are formed by a displaceable piston body within the first chamber, the method further comprising a centrifugation of the device about the common axis to substantially separate the blood in a cell fraction and a plasma fraction followed by the resulting plasma fraction is transferred while being influenced by the piston body to a second defined chamber by an outer cylindrical wall, which extends coaxially with the common axis, thereby a fraction with non-crosslinked fibrin polymer is caused to separate in the second chamber while a suitable enzyme is being added. This method is characterized in that the fraction of plasma containing fibrinogen is subjected to the enzyme during centrifugation, so that the resulting non-crosslinked fibrin polymer is deposited on the cylindrical outer wall of the second chamber, subsequently, the fraction of fluid collected at the bottom of the second chamber is transferred while being influenced by the piston body to the first chamber, and that the fraction with the non-crosslinked fibrin polymer remaining in the second chamber deposited substantially on the cylindrical wall is caused which dissolves by the addition of a solvent and by centrifugal stirring. Subsequently, the enzyme can be removed if desired, and the fibrin monomer solution produced in this way is transferred to any desired recipient vessel. Therefore, an aseptic condition is easily maintained to collect the solution. After the fibrin monomer has been redissolved, it can be transferred to an industry container, such as a syringe for further use as described in the prior art. Before transfer, the enzyme can be removed by any convenient means. The above copending application further describes a device for separating the components of a liquid by means of centrifugation around a central axis of rotation comprising a first annular chamber defined by an outer cylindrical wall and by an inner cylindrical wall, both walls being they arrange concentrically around the axis of rotation, as well as by a wall of the upper part and a bottom wall, where the bottom wall is formed by a displaceable piston body within the first chamber, the device also comprising a second chamber which communicates with the first chamber through a first conduit and which is defined by an outer cylindrical wall arranged concentrically around the axis of rotation and by the piston body and a bottom wall, where the second chamber is adapted to be placed below the first chamber during centrifugation, and where the device also comprises a gave blood feed to feed blood to the first chamber and a composition feeding means to feed composition that promotes separation as well as a receiving means for the collection of at least one liquid receiving container, where the receiving medium communicates with the second chamber through a second conduit. In a preferred embodiment, the piston rod comprises the inner chamber of the first chamber. This inventive device for carrying out the second method with the copending invention is characterized in that the first conduit comprises at least one channel extending between an opening in the wall of the upper part of the first chamber and an opening in the wall of the first chamber. background of the second camera. As a result, a device is provided which is relatively simple and which, independent of the position of the piston, ensures an easy and rapid transfer of the fractions in question from one chamber to the other chamber, and especially of the fraction of fluid from the second chamber to the chamber. the first chamber after the separation of the separation of the fraction containing fibrin I. The latter is especially due to the fact that the fluid is automatically concentrated at the bottom of the second chamber when centrifugation is stopped, with which it can be transferred easily to the first chamber by the moving piston.

According to the copending application, it is particularly preferred that at least one channel extends through the interior of the outer cylindrical wall in both the first and the second chamber with the result that the device is particularly simple and easy to use. In addition, the channel opening in the bottom wall of the second chamber can be centrally accommodated in the chamber in connection with a depression formed by the bottom wall. As a result, the fluid fraction in question is easily and rapidly guided directly to the inlet opening of the channel. Alternatively, each channel can be formed by a tube extending rectilinearly through the body of the piston and which is secured at the ends on the wall of the upper part of the first chamber and the bottom wall, respectively, of the second chamber where it communicates with the portions of the channel that end in the respective chamber. Additionally, the first chamber and the second chamber can in a particularly simple manner comprise a common outer cylindrical wall formed by an outer cylinder and an inner cylinder that hermetically fit into one another defining between them an axially extending channel, and cylinders can be terminated at one end by a terminal wall comprising an opening that allows the passage of a piston rod connected to the piston body, the piston body that forms the bottom wall of the first chamber and separates the first chamber of the second chamber, and where the channel extends between the end walls of the cylinders to an opening adjacent immediately to the piston rod. In the use of this device and method, suitable reagents are preloaded in the second chamber to facilitate the separation and treatment of the desired components within the blood plasma. For example, EP 592,242 discloses that the biotin-avidin capture system can be conveniently used to remove batroxobin from the desired solution. The biotin batroxobin is required to be present in the second chamber to react with the fibrinogen within the plasma and convert it to a fibrin monomer (which is immediately converted to a fibrin polymer). In order to subsequently capture the biotinylated batroxobin using the biotin-avidin system, the avidin which is bound, for example, the agarose must be present in the second chamber. In a closed, automated centrifuge device, these agents need to be loaded into the device before the blood is processed. The preload of biotinylated batroxobin and avidin-agarose in the same chamber has provided difficulties since the high affinity of avidin for biotin, which depends on the capture of the enzyme, prevents sufficient quantities of the enzyme from the first reaction with fibrinogen as required. A second co-pending application filed concurrently with the present entitled "Centrifuge With Annular Filter" discloses a device that makes it possible to easily place one or more reagents within a reaction chamber and release these reagents in a desired sequence. Preferably, when used in a device of the type described in the copending application mentioned above, reagents such as an enzyme and an enzyme capture composition can be released as desired. In satisfying the above object, according to the invention, there is provided a device characterized in that a capsule is accommodated in the second chamber and comprises a plurality of compartments for receiving respective compositions that promote separation, and that the capsule comprises a closing means that closes the compartments and while being influenced by the piston that adapts in sequence to the opening for the release of the contents of the compartments. This capsule makes it possible in a simple and easy way to feed the substances necessary for the separation of fibrin I, this capsule is preferably provided with these substances in advance. In addition, the compartments provided allow a predetermined, uniform distribution of the quantity in question. Batroxobine is preferably placed in a compartment in chemical relation to biotin with the proviso that the batroxobin enzyme can be easily captured after use by means of avidin, which is therefore placed in the second compartment in chemical relation with the agarose in the form of relatively large particles. The high affinity of biotin for avidin provides that the batroxobin and biotinylated / avidin-agarose particles, complex, are subsequently removed easily by filtration of the fibrin I solution. The placement of the two substances in their respective compartments makes It is also possible to easily dose the substances at the desired times due to the influence of the piston. The above substances or compositions, biotin-batroxobin, respectively and avidin-agarose can be used in any convenient way, for example, in the form of lyophilized powder. In accordance with the reagent distribution system of the copending invention, it is particularly preferred that the capsule comprises a central axis coaxially mounted inside the second chamber and carrying three mutually spaced radial disks forming divisions in the compartments and they are of an outer circumferential contour, substantially identical, and the closure means is formed by a displaceable sleeve-like body, but which radially surrounds the radial discs.To activate the displaceable body, in the form of a sleeve, the piston can comprise according to the invention, advantageously, a flange had downwardly cooperating with the sleeve-shaped body in the capsule to move the body in the form of a sleeve step by step whereby in sequence it opens for release of the contents of the capsules. compartments within the capsule According to the copending invention of the distribution system of ective, the capsule can be accommodated in connection, with an axial passage to a third adjacent chamber, the outer side of the capsule-shaped body of the capsule abutting hermetically with the side wall of the axial passage at least after an initial displacement of the body, whereby the lowermost division of the capsule allows a free passage of the liquid from the second chamber to the third chamber after the final displacement of the sleeve-like body caused by the piston-outlet of its coup with the circumference of the body. the lowest division. In this way, the capsule additionally forms an advantageous part of the device and aids the device in its additional operation during the separation of fibrin I. Additionally, to form an integrated part of the device, the center of the capsule may comprise in accordance With the invention, an axial pass passageway is secured in an upward projection, placed centrally at the bottom of the third, lower chamber, this pass passage in the bottom with communication for liquids with the outer annular compartment of the third chamber through a channel system, and the upper end of the center can be adapted to be hermetically connected to an axial passage in the piston body to be connected to a liquid receiving container insurable thereto. To remove one or more reagents from the desired solution of the product, the resulting fraction of fluid containing fibrin I is placed in a syringe through a filter while being influenced by the piston. As a result, the fibrin I solution is forced through the filter while the enzyme and other substances mixed to accelerate the separation are retained by the filter. The resulting performance of fibrin I is, however, not completely satisfactory when compared to the amount of fibrin I present in a blood sample.

BRIEF DESCRIPTION OF THE INVENTION Therefore, an object of the invention is to provide a method that makes it possible to achieve a superior performance of fibrin I by means of a device of the treated type. In the satisfaction of the above object, there is provided, according to the invention, a method characterized in that the portion of the fraction of liquid remaining in the second chamber before transfer to the liquid receiving container is transferred to a second one. chamber accommodated coaxially with the other chambers, and that the liquid now present in this third chamber is caused to pass through an annular filter during centrifugation to enter an outer, annular compartment that is adapted to be connected to the liquid receptor member . As a result, the fibrin I solution can be passed through a filter under the influence of the centrifugal force which is more considerably effective than the filtration by means of the piston. The invention also relates to a device for carrying out the above method. The inventive device comprises a first annular chamber defined by an outer cylindrical wall and an inner cylindrical wall, both walls which are arranged concentrically around the axis of rotation, and by a wall of the upper part and a bottom wall, where the upper part or the bottom wall is formed by a displaceable piston body within the first chamber, the device further comprising a second chamber communicating with the first chamber through a first conduit and defined by an exterior cylindrical wall accommodated concentrically about the axis of rotation, the bottom chamber of the first chamber and another bottom wall, where the second chamber is adapted to be placed below the first chamber during centrifugation, and where the device also comprises a feeding means of blood to feed blood to the first chamber and a composition feeding means for feeding com positions that promote separation as well as a receiving means for connecting at least one liquid receiving container, the receiving means communicating with the second chamber through a second conduit. According to the invention, this device is characterized in that the second conduit communicates with the second chamber through a third chamber accommodated coaxially relative to it and comprising a passage or passage to the second chamber that can be opened from the exterior, that the third chamber comprises an interior compartment and an exterior annular compartment, the compartments that are interconnected through a radially extending circumferential passage, in which an annular filter is arranged to prevent the passage of the fluid containing ingredients not desired, used to promote separation.

According to the invention, the passage between the second chamber and the third chamber can be arranged coaxially with respect to the two chambers and is closed by means of a capsule which is described in the copending patent application, mentioned above, "Centrifuge Reagent Delivery System ". This capsule comprises a central hub mounted coaxially in the second chamber and carrying or carrying a plurality of separate radial discs that form partitions in a plurality of compartments in the capsule, where the discs are of an identical outer circumferential contour, which outside the compartments are closed by means of a sleeve-shaped body, movably mounted, seal-like, the outer side of which is adapted to hermetically adjoin the side wall of the axial passage, the disc forming the division, lowermost , of the capsule that allows a free passage of liquid from the second chamber to the third chamber by an axial displacement of the sleeve-shaped body out of its engagement with the circumference of the lowermost division while being influenced by the piston. As a result, an easy and simple access to the filter in question is obtained by means of a capsule which is already used to feed the substances necessary to promote the separation of the fibrin I inside the second chamber. This capsule is preferably activated by the piston body comprising a downward flange which extends coaxially with the sleeve-shaped body of the capsule and which is adapted to engage the body when the piston is pressed downward to thereby open in sequence at appropriate times for the respective compartments in the capsule and finally to open the fluid passage from the second chamber to the third chamber. In order to facilitate the transfer of the fibrin solution I to the recipient vessel of the liquid, such as a syringe, the hub or center of the capsule may comprise, according to the invention, an axial passageway and is secured in a projection towards top arranged centrally in the bottom of the third lower chamber, this passage passage having communication with liquids with the outer annular compartment of the third chamber through a channel system, and the upper end of the hub or center can be adapted to to be connected hermetically to a passage or passage in the piston body to be connected with a liquid receiving container insurable thereto.

Finally, according to the invention, the hub or center of the capsule can be secured to the projection at the bottom of the third chamber by means of a sleeve, which at each end surrounds the tube and the projection, respectively, and the sleeve it may comprise a circumferentially projecting, outwardly projecting wall portion, whereby the annular filter is secured between the outer circumference of the wall surface and the bottom wall of the second chamber, whereby the portion of wall that comes out outwardly from the sleeve accommodates a distance from the bottom of the third chamber and thus forms the connection between the outer annular compartment and a channel extending axially in connection with the pass passage of the hub or center and extending between the outer side of the projection and the adjacent inner side of the sleeve. The resulting device is particularly simple. The methods of the present invention deal with improved processes for separating and isolating an individual blood component or a solution containing this component. However, the present method is suitable for any process adaptable to a cylindrical centrifuge, wherein a first solution is treated with one or more catalysts or reagents during centrifugation. Other blood procedures that could benefit from this method include, but are not limited to the isolation of any blood component, such as platelet-rich plasma, platelet concentrate, cryoprecipitate fibrinogen, other proteins within the plasma such as thrombin, fibronectin and the like. Preferably, the blood is an individual donor and in the most preferred form the blood is from the same person to whom the blood component will be administered. While the present methods are described hereinafter in terms of producing a solution of the fibrin monomer, the scope of the invention as will be appreciated by those skilled in the art, should not be limited in this way. As used herein, the term "centrifugal agitation" refers to the movement of the device where the redissolving buffer solution is introduced to redissolve the intermediate product such as non-crosslinked fibrin polymer gel, from the walls of the outer chamber . This centrifugal stirring or movement may include centrifugation to ensure that all of the exposed surface area of the gel is subjected to the redissolution solution, and preferably includes a centrifugation followed by stop and start rotations in the same direction and / or stop and start rotations. in opposite directions. Typical centrifugal agitations include, but are not limited to, turns of 5-30 seconds, preferably turns of 5-10 seconds, at 2,000-5,000 RPM in forward / backward cycles, repeated for any desired duration of time. In the present methods, turns of 5-10 seconds at about 3,000 RPM in forward / backward cycles, repeated for 1-2 minutes, are preferred. As mentioned above, this can be continued by a somewhat longer turn, for example, 20 seconds or more to initially distribute the solvent. The term "fibrin" as used herein refers to fibrin I, fibrin II or desßß fibrin. The present device incorporating the annular, centrifugal filter system described herein provides an efficient and accurate method for recovering one or more reagents from a desired product solution. This is especially critical in automated, self-contained, closed centrifuges for use in blood separation techniques, where it is required that two or more reagents be introduced into a reaction chamber in a sequential manner and therefore removed. In the preferred methods and devices described herein to provide a solution containing fibrin monomers, for example, for use in a new fibrin sealant, the sequential introduction of biotinylated batroxobin followed by avidin-agarose into the chamber that contains plasma provides a highly sophisticated method to prepare this solution.

BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present device and methods will now be described with reference to the drawings, in which Figure 1 is a sectional, axial view through a preferred embodiment of a device according to the invention, and Figure 2 illustrates a second embodiment of the device according to the invention.

The present device is an automated, closed, individual device, capable of converting whole blood into the desired blood components, preferably, components derived from themselves, useful, for example, as fibrin sealants.

DESCRIPTION OF THE PREFERRED MODALITIES OF THE PRESENT INVENTION Preferably, the present centrifugal, annular filter system is employed with a device as covered in the above copending, referred applications and is therefore described below with response to this device. However, it should be understood that it could be used in any reaction chamber device that requires the removal of one or more reagents. The device of Figure 1 according to the invention is constituted of parts which substantially have a rotational symmetry and which imply that the device can be placed in a centrifuge apparatus in an easy manner known per se to be centrifuged around an axis 1. In this Figure 1, a preferred embodiment of the device comprises an outer container 2 and an inner container 3 which are such that they completely fit together and adjoin each other closely from the portion where an intermediate channel 4 is provided. It extends axially. The channel 4 is provided by a slot formed in the inner container 3. The two containers 2 and 3 comprise their respective upper portions 5 and 6, respectively, defining a central opening 7 that allows the passage of a rod 8. of piston. Around the opening 7, the two containers comprise the parts 9 and 10, which extend axially, respectively, which extend close to the piston rod 8, hollow in a direction away from the interior of the containers. The outer container 2 abuts the hollow piston rod along a radially extending, short rim 11 provided with a depression 12 that receives a seal ring 13. As illustrated in Figure 1, the channel 4 continues between the inner container and the outer container all the way from the outer cylindrical walls of the inner container and the outer container along the portions 5 and 6 of the upper part and the axial parts 9 and 10 to the opening immediately below the seal ring 13 in the opening 7. The axial part 10 of the inner container 3 abutting the opening 7 is dimensioned so that there is a narrow, but free passage towards the interior of the leads 2 and 3 around the piston rod 8, hollow. The outer container 2 comprises a cylindrical part of a uniform diameter, compare Figure 1. Downward, when seen in relation to the drawing, this part continues to a cylindrical part 14 of a slightly larger diameter through a transition part 15, short that forms an interior surface, frustoconical. The inner container 3 ends at the location where the transition part 15 the outer container 2 continues to the cylindrical part 14 of a larger diameter. The lower end of inner container 3 comprises an outer surface 17 of a frusto-conical shape that matches the shape of the frusto-conical surface 16 on the inner side of outer container 2. An outer and inner annular disc 19 and 20 are provided, respectively, immediately below the lower end of the inner container 3, which ends at the radial surface 18. These discs abut closely together, apart from the fact that they define between them a channel 21 extending in an axial plane from a central opening 22 and towards the inner side of the outer container 2, wherein the channel 21 communicates with the channel 4 between the outer container 2 and the inner container 3 through the axially extending part 23. The channel 21 and the axial channel portion 23 are suitably provided by means of a groove on the side of the inner disc 20 which gives the outer disc 19. The two discs 19 and 20 are formed with this oblique course comprising substantially fruit-conical interior surfaces. and outer, compare Figure 1, and thus tilted downward towards the central opening 22. Figure 1 also shows that the inner disk 20 comprises a radial surface 24 abutting the radial surface 18, adjacent, in the inner container 3. The radial surface 24 of the inner disk 20 is provided with a depression 25 for receiving a seal ring 26. The two discs 19 and 20 are held in their abutting position against the radial surface 18 of the inner container 3 by means of a cover 27 which closes the outer container in a downward direction. This cover 27 covers a circumferential sleeve-shaped part 28 adapted to closely abut the inner side of the outer container 2, to which it is suitably secured, such as by means of a snap closure action by the coupling between a circumferential rib 29 on the outer side of the sleeve 28 and a corresponding circumferential groove 30 on the inner side of the outer container 2. A connection of the seal is secured by means of a seal ring 31 in a circumferential depression 32 in the outer periphery of the outer disk 19. The cover 27 further comprises a relatively thin wall 32, adapted to form the bottom bottom of the device in the position shown in Figure 1. This wall 32 extends substantially along a course parallel to the disk 19 and 20, exterior and interior, in such a way that the wall 32 extends from the inner side of the sleeve 28 in an adjacent portion of the discs. 19 and 20 and down towards a portion substantially on one level with the lower edge 33 of the outer container 2. In order to reinforce this relatively thin wall 32, a radial, reinforcement rib 34 is provided at regular intervals, only one of the ribs shown in Figure 1. This rib 34 is partially formed with a portion positioned outside the wall 32 and partially with a portion placed inside the wall 32, compare Figure 1. This last portion is designated, with the number of reference 35 and such that it abuts the bottom side of the outer disc 19 with the result that it aids in the maintenance of the disks 19 and 20 in a reliable position. A dividing means 36 is tightened between the outer disc 19 and the cover 27. This dividing means 36 comprises a section 37 of the central tube. This section of tube is mounted on a tang 38 which projects axially inwardly and which is integral with the wall 32 of the cover 27. This tube section 37 is formed integral with a circumferential wall disc 39 that extends outwardly. from the tube section 37 in such a manner that it initially slopes downward slightly towards the wall 32 of the cover 27, after which it extends along a short axial course to continue in a course extending substantially parallel to the wall 32 of the cover. The wall disk 39 terminates in a radially extending, short periphery 40 that rests on a shoulder 41 in rib portions 35 in the cover 27. An annular filter unit 42 is applied between the outer periphery 40 of the disk 39 of wall and the bottom side of the outer disc 19. This annular filter unit 42 abuts a radially formed surface 43, substantially on the outer, adjacent side of the outer disk 19. A device and method employing this annular filter are the subject of a co-pending application filed concurrently with the present one entitled "centrifuge with Annular Filter". 'In order to ensure stability in the medium 36, are additionally accommodated between the tube section 37 and the wall disc 39, radial, reinforcing ribs designated by the reference numerals 44. The reagent distribution system of the present invention comprises a capsule designated by the number of general reference 45, which is secured at the opposite end of the cover 27 of the tube section 37 of the dividing means 36. This capsule is suitable for selectively releasing agents in the second chamber 75. This capsule comprises a section 46 of elongated tube formed integral with a radial ring 47 and carrying two additional radial rings 48 and 49. These radial rings 48 and 49 are secured by means of an interference fit on their respective side of the fixed ring 47. The loose rings 48 and 49 are accommodated in their respective distance from the fixed ring 47 by means of circumferential shoulders 50 and 51, respectively, in the pipe section 46. The three discs 47, 48 and 49 are all of the same outer diameter and carry along their respective peripheries a sleeve 52 mounted in a displaceable, circumferential manner. As illustrated in the drawing, the lower disc 49 abuts the upper end of the tube section 37 of the dividing means 36, whereby the positioning of the capsule 45 in the axial direction is determined. This position or positioning is further determined in such a way that when moving in the axial direction, the displaceable sleeve 52 of the capsule enters a seal coupling at its lower end, comparing the pattern, with the innermost edge 53 of the disc 19 outside in the central opening 22. In this position or positioning of the sleeve 52, there is still a communication between the space of the inner disk 20 surrounding the sleeve 52 and the entrance opening to the channel 21 between the outer disk 19 and the inner disk 20. The axial length of the displaceable sleeve 52 is adapted such that the engagement with the outer disc 20 occurs before the upper end, comparing the pattern, of the sleeve 52 decouples the fixed ring 47 during the downward, axial displacement of the sleeve 52. The inner diameter of the sleeve 52 also conforms to the outer diameter of the axially extending part of the wall disk 39 of the dividing means 36 in one order. such that a continuous, downward displacement of the sleeve 52 towards the cover 27 causes the sleeve 52 to be fixedly atta to the dividing means 36 once the outer disc 19 has been decoupled. The length of the axial part of the dividing means 36 also corresponds to the axial length of the sleeve 52 in such a way that the sleeve 52 in the lowest position or position is substantially completely received by the dividing means 36. As illustrated in the drawing, the hollow piston rod 8 comprises a circumferential piston 55 inside the outer container 2 and the inner container 3, the piston 55 sealingly engaging the inner side of the inner container 3 through a ring 56 of seal. A Luer coupling 57 is formed within the hollow rod of the piston to receive a conventional syringe 58 with a piston-action cap 59 to act on the contents of the syringe 58. The coupling 57 is formed substantially as a length or section communicating. with a central opening 61 in the piston 55 through a frusto-conical portion 60. The tube section 57 is provided with a web 62 that projects radially inward to direct the fluid leaving the syringe 58 away from an axial path and thereby surround the length of the tube 46 below it within the capsule 45. This last tube section 46 is of such length and dimensions that it can be hermetically coupled with the length or section of the tube 57 inside the piston rod 8, hollow when the piston 55 is in its lowermost position near the cover 27. In order to promote the airtight connection above, the inner side of the tube section 57 is formed with a diameter gradually decreasing at the end adjacent the piston 55. An axially projecting flange 63 is formed integral with the piston 55 around the central opening 61 of the piston. The flange 63 is formed with a diameter of such a length that by a suitable displacement of the piston 55 it can activate the forward displacement of the displaceable sleeve 52 of the capsule 45 in the positions in which the inner edge 53 of the central opening engages 22 through the two rings 19 and 20 followed by a coupling of the dividing means 36. An annular, resilient lip seal means 64 is secured as indicated around the hollow piston at the top inside the containers 2 and 3, comparing Figure 1. This lip seal means 64 is adapted to prevent undesired passage of fluid from the interior of the containers 2 and 3 towards the channel 4, but allows the passage of the fluid when a force is applied through the piston 55. As indicated in the upper part of Figure 1, a connection to the fluid is provided. a hose 65 through an opening 66 in the outer and inner container 2 and 3, respectively. This connection is not known and therefore is not shown in greater detail, but allows an interruption of the connection to the hose when desired. In addition, an air exhaust opening with a suitable filter is provided in a conventional manner and therefore neither shown nor described in greater detail. A passage 69 is provided from the area between the dividing means 36 and the cover 27 and all the way up through the inside of the length of the tube 37 of the dividing means 36 and through the inside of the length of the tube. 46 of the capsule 45. This passage or passage 69 allows a transfer of the fluid to the syringe 58 from the area where the latter length of the tube 46 fits the length of the tube 57 inside the piston rod 8. The passage or passage 66 is provided in the lowermost portion of the tang 38 in the cover 27 by the tang 38 that is formed with an axial, flat surface, the shank being of a substantially circular cross section. As a result, a space is provided between the peg and the adjacent portion of the inner side of the length of the tube 37. An area 67 is provided and immediately above the peg 38 where the dividing means 36 has a slightly reduced inner diameter. In this way, it is possible to place a small filter 68 immediately above this area, compare Figure 1, whereby the fluid must pass the filter before it enters the length of the tube 46 of the capsule 45. The described device comprises a first annular chamber 70 defined inwardly by the hollow piston 8 forming an internal, cylindrical wall 71, and outwardly by an outer, cylindrical wall 27 formed by the outer container 2 and the inner container 3. When in the position of conventional use, compare Figure 1, the annular chamber 70 is defined upwards by a wall 73 of the upper part formed by the bottom 5 and 6, respectively, of the outer container 2 and the inner container 3. Downwards, the annular chamber 70 is defined by a wall 74 of the bottom formed by the piston 55. A second chamber 75 is defined below the piston 55, the second chamber downward which is defined by the same exterior wall 72, cylindrical as the first chamber 70. Downwardly, the second chamber 75 is defined by a second wall 76 of the bottom formed by the outer disc 19 and the inner disc 20. The capsule 45 is centrally accommodated within the second chamber 75. A third chamber 77 below the second wall 76 of the bottom, and this third chamber 77 is defined by the dividing means 36 and the annular filter unit 42. In addition, this third chamber 77 communicates with the second chamber 75 through the passage or passage formed by the central opening 22 in the outer disc 19 and the inner disc 20. Finally, a fourth chamber 78 is provided below the dividing means 36, this fourth chamber 78 is defined downwardly by the wall 32 of the cover 27 and additionally by portions of the sleeve 28 of the cover 27 and the bottom side of the disc 19. As described above, the described device is mainly suitable for the separation of a component, such as fibrin monomer from blood, and for this purpose, the second chamber, 75, and preferably the upper chamber 80 of the capsule 46 is filled in advance with a suitable enzyme, such as batroxobin. As understood from EP-PS No. 595,242, any enzyme similar to thrombin can be extended. These enzymes include thrombin itself or any other material with similar activity, such as Ancrod, Acutin, Veniimin, Asperase, Botropase, Crotabasa, Flavorxobin, Gabonasa, and the preferred Batroxobin. Batroxobin can be chemically linked to biotin, which is a synthetic substance that allows batroxobin to be captured in a conventionally known manner by means of avidin in an avidin-agarose composition. Accordingly, avidin-agarose is found in the lowermost chamber 81 of the capsule. Both the biotin-batroxobin composition and the avidin-agarose composition are relatively easy to fill in the respective chambers 80 and 81 within the capsule 45 before the capsule is placed inside the device.

Finally, a syringe 58, the syringe containing a pH 4 buffer prepared from an acetate diluted with acetic acid and suitable for receiving fibrin I, is arranged. Any known prior art cushion can also be used. The redissolving buffer may be any acid buffering solution, preferably those having a pH between 1 and 5. Suitable examples include acetic acid, succinic acid, gluconic acid, cysteic acid, crotonic acid, itaconic acid, gluonic acid, formic acid, aspartic acid, adipic acid, and salts of any of these. Succinic acid, aspartic acid, adipic acid and salts of acetic acid, for example, sodium acetate are preferred. Also, the solubilization can be carried out at a neutral pH by means of a cauteric agent. Suitable agents include urea, sodium bromide, guanidine hydrochloride, CKNS, potassium iodide and potassium bromide. The concentrations and volumes of these acidic buffers or that chaotropic agent are as described in EP-PS No. 592,242. During or immediately after the blood supply, the rod 8 of the piston is pushed into the interior of the device whereby the displaceable sleeve 52 of the capsule 45 moves downwards in a seal coupling in the pass passage through the blood. the bottom wall 76 and the second chamber 77. As a result, access to the biotin-batroxobin composition is opened simultaneously within the uppermost chamber 80 of the capsule. When the device is ready for use, a blood sample is fed into a first chamber through a needle not shown and the hose 65 in a conventional manner, the blood sample that is mixed with an anticoagulant also in a conventional manner . During the limitation of the blood through the hose 65 and the opening 66 in the interior in the first chamber 70, the air is removed from the chamber in a conventional manner. After the blood supply, the hose 65 is removed, and the opening 66 is hermetically sealed. Subsequently, the device with the blood is placed in a centrifuge that helps inter alia in the hermetic compression of the various parts. The centrifuge causes the device to rotate about the axis of rotation 1. As a result of centrifugation, the blood is separated in the first chamber 70 into a plasma fraction that sits radially within the remaining portion of the blood, this portion remaining It contains red blood cells and white blood cells. As described in EP-PS No. 592,242, platelets can be present in any fraction, as desired, by varying the speed and time of centrifugation. When it is between the plasma and the remaining portion of the blood, it has stabilized, that is, when the separation is completed, a reduction in the volume of the first chamber 70 is initiated by the rod 8 of the piston and consequently the piston 55 pull. As a result, a possible inner air layer passes through the channels 4 and 21 towards the second chamber 75, and an additional movement of the piston 55 implies that also the plasma passes into the second chamber 75. The movement of the piston 55 for when the entire plasma layer has been forced into the second chamber 75, that is, when the face between the plasma fraction and the remaining portion of the blood has reached the inner wall 71 of the first chamber 70. In the second chamber 75, the plasma fraction comes into contact with the bactroxobin enzyme with the result that the fibrin monomer, which polymerizes immediately to a non-crosslinked fibrin monomer, is released from the plasma fraction. This process is performed while the device is being continuously centrifuged with the result that the fibrin polymer efficiently separates from the resulting portion of the plasma fraction. This fibrin polymer that is formed by the reaction of the biotin-batroxobin composition and which sits as a viscous layer along the outer cylindrical wall 72. When this separation is over, separates the centrifugation, whereby the remaining relatively fluid portion of the plasma fraction can be easily pressed back to the first chamber 70 by the piston 55 which rises to transfer the air from the first chamber 70 to the second chamber 75 followed by the piston 55 that is pressed down. This transfer can be carried out relatively easily and quickly before the viscous layer with the fibrin polymer reaches the opening towards the channel 21. Optionally, additional measures can be taken in order to prevent the viscous layer from reaching the entrance of the channel 21 too much. rapidly, such as by providing a ring of teeth 82 projecting upwardly shown by discontinuous lines at the bottom 76. This spin / fill method can be carried out two or more times, as may be required, to obtain both Plasma fluids of the fibrin polymer as possible. Once the remaining portion of the plasma fraction has been expelled from the second chamber 75, the displaceable sleeve 52 of the capsule 45 is further moved downward in a manner such that access to the lowermost chamber 81 is allowed. At the same time, or in conjunction with the last displacement of the sleeve, the plug 49 of the syringe 58 is pressed completely downwards by means of a spindle acting from the outside in such a way that the buffer pH 4 is transferred to the second chamber 75, which can be done while centrifugal stirring is initiated. The addition of the pH 4 buffer is provided that the fibrin polymer is dissolved therein, and the presence of the avidin-agarose composition in the lower chamber 81 within the capsule 45 is provided that the composition of biotidine-batroxobin is bound in a conventional manner by avidin. A continuous movement of the piston 55 causes the sleeve 52 displaceable in the capsule 45 to engage the dividing means 36 and to uncouple the wall 76 from the bottom with the result that free access is provided to the third chamber 77. As a result, the contents of the second chamber 75 can flow freely down to the third chamber 77. Preferably, the redissolution is carried out during the centrifugal stirring comprising the centrifugation and a series of stops and starts of agitation movements forward / forward behind. A continuous centrifugation is provided that the fibrin monomer solution can be separated in the third chamber through the annular filter unit 42 which retains the relatively large agarose particles and the batroxobin bound thereto via the capture system biotin-avidin. When the solution of the fibrin monomer has passed to the fourth lowermost chamber 78 as a result of the above centrifugation, the centrifugation is stopped and the fibrin solution I is easily transferred to the syringe 58 by a renewed feedback of the plug 59, the end more superior of the length or section 46 of the capsule 45 which couples the length of the tube 47 that forms the connection with the syringe 58. As the fibrin polymer is separated from the plasma fraction in the second chamber 75, during a continuous centrifugation and as 1 fibrin monomer solution is separated in the third chamber 77, on centrifugation it is possible to achieve a relatively high yield of fibrin I from the blood sample in question. The invention has been described with reference to a preferred embodiment. However, many modifications can be made without departing from the scope of the invention in this way. Figure 2 illustrates examples of these modifications, as Figure 2 illustrates a second embodiment of the invention corresponding more or less to the embodiment of the invention shown in Figure 1. The embodiment of Figure 2 comprises a first chamber 90 and a second chamber 91 separated by a piston 92, comprising a hollow piston rod 93 defining the first chamber inwards. Outside, the two chambers are defined by a portion of a substantially tubular member 94 forming and the outer cylindrical wall 95 for the two chambers 90 and 91. Upwardly, the first chamber 90 is defined by a wall 85 of the top portion that a in turn, it is formed by a cover of the upper part secured to the tubular member 94 by means of a ring 96 screwed into the tubular member 94. The wall 85 of the upper part defines a passage opening for the passage of the hollow piston rod 93. Downwardly, the second chamber 91 is defined by a bottom wall 96 formed by a circumferential inner rim in the tubular member 94. On the side adjacent the second chamber 91, the tubular member 94 comprises a frustoconical surface 97 that slopes away from the piston 92 towards the center of the second chamber 91. The bottom wall 96 defines a central passage 98 through a third chamber 99. The third chamber 99 is defined by a separation means 100 and an annular filter unit 101. inserted between the bottom wall 96 and the dividing means 100 and leading to the fourth annular chamber 102. The fourth chamber 102 defines between a cup-shaped cover 103 secured to the tubular member 94 by threads. The cover 103 is retained through the intermediate ribs 103 to the dividing means 100 in its position centrally within the tubular member 94 while tightening the annular filter unit 101. A capsule 105 is secured in a spigot 104 projecting centrally and upwardly in the dividing means 100. The capsule 105 comprises a tubular portion 106 with disc-shaped rings 107, 108 loosely attached thereto and defining a chamber for these enzymes indicated by the letters BB and AA, respectively, by means of a displaceably arranged sleeve. The disc-shaped rings are secured at desired mutual distances in the length of the tube 106 by means of ridges formed therein by the outer periphery of the tubular member 106 which is a diameter descending from below and upwards. The passage channels 115 and 116 are provided from the top of the first chamber 90 to the bottom of the second chamber 91. These channels are provided by means of their respective fixed length of the tube 117 and 118, respectively, which extend parallel to the axis of rotation of the device and securing the ends in associated openings in the wall 95 of the upper part and the wall 96 of the bottom. The connection of the channels between these lengths of the tube and the chambers, respectively, is provided by suitable holes and plugs secured therein. The tube lengths 117 and 118 extend through their respective openings in the piston 92. Seal rings are provided anywhere to prevent leakage. A coupling 120 is centrally secured within the piston 92 for coupling to a syringe 121 within the hollow piston rod 93 and to the upper end of the length 106 of the capsule tube 105. The coupling 120 carries a flange 122 and projects to the second chamber 91 and which influences the sleeve 110 movable in the capsule 105. As illustrated, the outer diameter of the sleeve 110 fits the diameter of the passage 98 downwardly towards the third chamber 99 in such a way that the sleeve 110 is guided and stopped by the wall 96 of the bottom in any position and consequently also in a lowermost position in which the sleeve 105 does not engage the ring 109 in the form of a disc, more lower in the capsule and allows the passage of flow from the second chamber 91 to the third chamber 99. A channel 123 extends from the fourth chamber 102 and passes centrally upwardly through the spike 104 in the dividing means 100 and then further up through the tubular member 106 of the capsule 105, fluid is allowed to enter the syringe 121 therefrom. The device of Figure 2 is used in a completely the same way as the device of Figure 1, whereby means are also provided, of course, for attaching a hose thereto for the blood supply.

The described parts forming part of the various devices are easily manufactured from suitable plastic materials by means of injection molding, and the devices in question are therefore also relatively inexpensive and suitable for disposable use. The invention has been described with reference to the preferred embodiments of the device. However, the method according to the invention can be easily carried out in a laboratory under aseptic conditions by means of a layer which is closed by a lip. Plasma and enzyme are filled into the cup and upon mixing and following centrifugation, the non-crosslinked fibrin polymer is removed at the bottom or the wall of the cup as described above. After removal of the remaining plasma fraction, the non-crosslinked fibrin polymer is redissolved by the addition of a solvent and by centrifugal stirring as described above as well.

EXAMPLE 140 ml of whole blood and 20 ml of sodium nitrate anticoagulant (USP) were introduced into the first chamber 70 of the device described above. This combination was centrifuged for 2 minutes at approximately 6,000 RPM to provide separation of plasma and blood cells. While centrifugation is continued for mainly separation, the piston rises for transfer to the innermost phase, ie, the plasma, in the second chamber 75. Approximately 60 ml of plasma was transferred. This was treated with 30 units of biotenylated batoxobine which was introduced into the second chamber 75 via, the chamber 80 of the capsule 45 as previously described. Plasma and batoxobine were mixed at a slow speed, for example, approximately 2,000 to 3,000 RPM and then centrifuged for 9 minutes at 9,000 RPM. The non-crosslinked fibrin polymer gel was precipitated as a thin gel layer on the cylinder walls and rotation was stopped. The remaining plasma fluid (serum) was then transferred back to the first chamber 70. This was followed by two additional one minute centrifugation at 9,000 RPM to remove as much serum from the gel as possible. After each 1 minute centrifugation, excess serum was transferred to the first chamber 70.

Subsequently, a buffer solution comprising 3.5 ml of 0.2 M sodium acetate (pH 4) containing 24 mM calcium chloride was introduced from the second chamber 75 via the syringe 58. At the same time, a centrifugal stirring comprising turns of 5-10 seconds at about 3,000 RPM each in repeated forward / backward cycles was carried out for 2 minutes to dissolve the fibrin polymer gel and provide a solution containing fibrin monomer. To the separate solution was added avidin-agarose via the lower chamber 71 of the capsule 45. This was followed by an additional centrifugal stirring consisting of turns of 5-10 seconds at about 3,000 RPM in repeated forward / reverse cycles for 5 seconds. minutes The resulting solution with fibrin monomer tube plus an avidin-agarose complex: biotin-batroxobin. This solution was transferred to the third chamber 77 and filtered with centrifugation through a Porex annular filter, 20 μm for 1 minute at 9,000 RPM. The resulting solution of the fibrin monomer was collected in the syringe 58 as previously described. The fibrin monomer solution formed in this manner (fibrin I in this case) was re-polymerized in a fibrin sealant by co-administration to a site in need of this sealant with a 0.75 M sodium carbonate / bicarbonate buffer. at a ratio of fibrin I: 5: 1 buffer It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (6)

1. A method for separating a component, such as fibrin I from blood, by centrifugation, the method comprising feeding blood mixed with the anticoagulator to a first annular chamber in a device, wherein the annular chamber is defined by a cylindrical outer wall and a inner cylindrical wall, both walls that extend coaxially around a common axis, as well as by a wall of the upper part and a bottom wall, where the wall of the upper part or the bottom wall is formed by a piston body movable within the first chamber, the method further comprising a centrifugation of the device about the common axis followed by a fraction of the resulting liquid that is transferred while being influenced by the piston body to a second chamber defined by an outer cylindrical wall, which extends coaxially with the common axis, whereby the liquid fraction present in the second chamber is caused to be further separated by a continuous centrifugation and the addition of suitable compositions that promote the separation and return of the usable portion to the first chamber, and whereby the portion of the liquid fraction remaining in the second chamber is transferred to a recipient vessel of liquid through a filter optionally after the addition of a solvent, characterized in that the portion of the liquid fraction remaining in the second chamber before transfer to the liquid receiving container is transferred to a third chamber accommodated coaxially with the other cameras, and in that the liquid now present in the third chamber is caused to pass through an annular filter during centrifugation to introduce an outer, annular compartment that is adapted to be connected to the liquid receiving member.

2. A device for carrying out the method according to claim 1, and comprising a first annular chamber defined by an outer cylindrical wall and an inner cylindrical wall, both walls that are arranged concentrically around the axis of rotation, and by a wall of the upper part and a bottom wall, wherein the wall of the upper part or the bottom wall is formed by a displaceable piston body within the first chamber, the device further comprising a second chamber communicating with the first chamber through a first conduit and defined by an outer cylindrical wall arranged concentrically around the axis of rotation, the bottom wall of the first chamber, and another bottom wall, where the second chamber is adapted to be placed by down the first chamber during centrifugation, and where the device also comprises a means of feeding blood to feed blood to the first chamber and a composition feeding means for feeding compositions that promote separation as well as a receiving means for the connection of at least one liquid receiving container, the receiving means communicating with the second chamber through a second conduit, characterized in that the second conduit has communication with the second chamber through a third chamber accommodated coaxially relative to it and comprising a passage to the second chamber that can be opened from the outside, in which the third chamber comprises a compartment interior and an outer annular compartment, the compartments interconnecting through a radially extending circumferential passage, in which an annular filter is arranged to prevent the passage of the liquid containing undesired ingredients to promote separation.

3. A device according to claim 2, characterized in that the passage between the second and third chamber is arranged coaxially with respect to the two chambers and is closed by means of a capsule, comprising a central hub mounted coaxially in the second chamber and which carries a plurality of mutually spaced radial disks that form partitions in a plurality of the compartments in the capsule, wherein the discs are of an identical, outer circumferential contour, in which outwardly the compartments are closed by means of a sleeve-shaped body, mounted in a displaceable, seal manner, the outer side of which is adapted to abut hermetically with the side wall of the axial passage in any of its positions, the disc that forms division, lowermost of the capsule that allows a free passage of liquid from the second chamber to the third chamber by an axial displacement of the body in the form of a sleeve between its coupling with the circumference of the lower division while being influenced by the piston.

4. A device according to claim 3, characterized in that the piston body comprises a downward flange which extends coaxially with the sleeve-shaped body of the capsule and which is adapted to engage with the body when the piston body is pressed down to thereby open in sequence at appropriate times the respective compartments in the capsule and to finally open the passage of fluid from the second chamber to the first chamber.

5. A device according to claim 3 or 4, characterized in that the cube of the capsule comprises an axial passage, and is secured in an upwardly arranged projection arranged centrally in the bottom of the third lower chamber, the passage passage that has communication for liquids with the outer annular compartment of the third chamber through a channel system, and in which the upper end of the hub is adapted to be hermetically connected to a passage in the piston body to be collected with a receiving vessel of the piston. insurable liquid thereof.

6. A device according to claim 5, characterized in that the hub of the capsule is secured to the projection of the bottom of the third chamber by means of a sleeve, which at each end surrounds the hub and the projection, respectively, in which the sleeve it comprises a wall portion projecting outwards, circumferentially, whereby the annular filter is secured between the outer circumference of the wall surface and the bottom wall of the second chamber, whereby the projecting wall portion outwardly from the sleeve accommodates a distance from the bottom of the third chamber and thus forms the connection to the outer annular compartment and a channel extending radially to the passage passage of the hub between the inner side of the projection and the side adjacent interior of the sleeve.