MXPA99006281A - Apparatus and methods for preparing blood or plasma component solutions of known concentration - Google Patents

Abstract

An apparatus for separating e.g., a fibrin monomer from blood includes a container (110) with a reaction chamber defined by an outer wall for receiving plasma and a means for supplying the chamber with an agent for converting the fibrinogen content of the plasma into a non-cross-linked fibrin polymer. The apparatus includes a device for centrifuging the plasma and the agent within the chamber to a degree sufficient for separating the fibrin polymer from the plasma, depositing the polymer on the outer wall of the chamber and expelling the remaining plasma from the chamber. The container (110) includes means for supplying the chamber with a solvent for dissolving the fibrin polymer.The apparatus includes a measuring device (130) for measuring the amount of the deposit of the fibrin polymer on the outer wall of the container (110) and a control unit (131) for controlling the addition of solvent in response to the amount of polymer.

Description

APPARATUS AND METHODS FOR THE PREPARATION OF PLASMA AND BLOOD COMPONENT SOLUTIONS FIELD OF THE INVENTION The present invention relates to an apparatus and methods for separating components, for example, fibrin monomer from blood or plasma. The invention further relates to such an apparatus and methods wherein the concentration of a solution resulting from such a component can be determined or controlled using sensors, for example, optical sensors.

BACKGROUND OF THE INVENTION International publication WO 96/16714 describes a container for separating a blood or plasma component, for example, fibrin monomer, from the blood or plasma by centrifugation about a vertical axis. This container comprises a first annular chamber defined by an external cylindrical wall and an internal cylindrical wall, both walls extending coaxially around an axis Re. : 30670 common, as well as also by an upper wall and a lower wall, wherein the lower wall is formed by a piston that moves inside the first chamber. The container further comprises a second chamber accommodated below the first chamber and communicating with the first chamber through a first conduit. The second chamber is defined by an external cylindrical wall, the lower wall of the first chamber, and by a second lower wall. This second chamber serves as a reaction chamber for receiving the plasma and treating the plasma to obtain the desired component. For example, treatment of plasma fibrinogen with thrombin or thrombin-like enzyme converts fibrinogen to fibrin monomer which spontaneously polymerizes to a non-crosslinked fibrin polymer. Placing this container in a centrifugal machine for the reaction described above provides that the non-crosslinked fibrin polymer is separated from the plasma and deposited on an outer wall of the reaction chamber during centrifugation. When the piston is actuated subsequently, the remaining plasma is removed from the reaction chamber. After this, a solvent is added to dissolve the non-crosslinked fibrin polymer thus deposited and the desired fibrin monomer solution is formed. As described in detail in EP 592242 this fibrin monomer solution is extremely useful, for example, in fibrin sealant methods. It is desirable to use devices similar to those described in U.S. 5,603,845, WO 96/16713, WO 96/16714 and WO 96/16715 for preparing blood products such as fibrin sealant components immediately at the same time as surgery so that blood derived from itself can be used. It may also be desirable from a surgeon's perspective to use sealing products which are relatively uniform from one procedure to another. This is clearly impossible for freshly prepared products, however, since the concentration of fibrinogen in human blood can vary by ± 300% in populations of human patients and freshly prepared sealant components from individual sources will also vary. More humans have fibrinogen levels between 2 and 6 mg / ml of plasma and some humans may have as little as 1 mg / ml and some as much as 10 mg / ml (fibrinogen plasma).

DESCRIPTION OF THE INVENTION . The object of the present invention is to provide an apparatus that allows a control of the supply of the solvent in response to the amount of a polymerized form of a desired component present in the reaction chamber. In satisfaction of the preceding object there is provided an apparatus which according to the invention comprises a measuring device for measuring the amount of the deposit of the polymerized component in the external wall, as well as a control unit for controlling the addition of the solvent in response what quantity of component. The measuring device or dispenser can be adapted according to the invention, advantageously, to continuously measure the quantity of the polymerized form of the desired plasma or blood component, at least immediately before and during the addition of the solvent. According to a particular embodiment, the measuring device can be, according to the invention, an optical device, and this optical device can be, according to the invention, a photometer.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is described in more detail below with reference to the accompanying drawings, in which FIGURE 1 is an axial sectional view of a container for separating fibrin monomer from blood plasma, FIGURE 2 is a view diagrammatic of an apparatus according to the invention during handling or handling of a container of the type shown in FIG. 1, FIGURE 2a is a second diagrammatic view of an apparatus in accordance with the present invention, and FIGURE 3 illustrates a graph showing measurements of a photometer against time.

DESCRIPTION OF A PREFERRED MODALITY OF THE PRESENT INVENTION The present invention provides an apparatus and methods for preparing solutions of a plasma or blood component of known or controlled concentrations. This provides the unique ability to prepare such solutions in an automated centrifuge unit for 30 minutes so that freshly prepared components, and preferably derivatives thereof, can be used. The possible disadvantage of using freshly prepared components or solutions, i.e., the fact that the levels of components may vary from patient to patient, is exceeded in the present invention. Not only can the concentration of a component solution, for example, a solution of fibrin monomers, be determined, but the amount of solvent or buffer used makes the solution controlable in response to this determination, so that it can be prepared any desired concentration. Essentially, the method and apparatus involves introducing blood or plasma into a container having a light transmitting wall and providing a reaction which results in a polymerized form of the component that is deposited on the wall. An optical reading of the difference of light transmission through the wall alone and the wall with the polymer therein can be related to the total amount of the component from the blood or plasma sample. Used in this manner, the present apparatus and methods are useful for determining the concentration of the component in the blood or plasma. In addition, knowing the amount of solvent or buffer that is used to solubilize the polymerized component to produce the solution of the desired component, the concentration of the resulting solution is easily obtainable. Still further, when the optical determination of the concentration of the component in the blood or plasma (or the determination of the amount of the polymerized component) is made, these data can be used to control the amount of buffer or solvent used to solubilize the polymer to prepare solutions of desired concentrations. Still further, when the buffer or solvent is also used so that the resulting solutions are of a specific value or range of pH values, limits may be used on the minimum and maximum amount of buffer or solvent used. For example, when a plasma fraction is reacted to form a fibrin polymer and an acetate buffer at pH 4 is used to solubilize the polymer to form a desired fibrin monomer solution, minimum and maximum amounts of buffer can be programmed. in the process and apparatus to maintain the resulting pH within a desired range, for example, 4.0-4.5. This, of course, places some limitations on the ability to make solutions of constant concentration of blood sources which have fibrinogen levels that vary ± 300% as in the human population. Even taking this limitation into consideration, the present methods can provide monomeric fibrin solutions of approximately 20 mg / ml ± 25% while simultaneously maintaining the pH value between 4.0-4.5. This represents a 10-fold remarkable increase in the reproducibility of the monomeric fibrin solution that is derived by itself or recently prepared. It is also important to note that the present optical sensor apparatus and methods are used to determine quantities / concentrations of components in a container that rotates at high speeds, for example, up to 9000-10,000 RPM rather than occupying an optical measurement in a fixed position. It was unexpected that such exact and reproducible data could be the result. Indeed, it is believed that a more accurate reading is obtained since the fast moving container provides more than one advantage of the present material. Throughout this application the present invention is described with respect to preferred embodiments, for example, preferred apparatus, containers and methods useful for preparing plasma fibrin or whole blood monomer solutions. However, it will be readily understood by those skilled in the art that other components of the blood or plasma may also be prepared or extracted using the general methods described herein.

The container of FIGURE 1 is known from the international application WO 96/16714 above and is constructed of parts that mainly have a symmetrical rotation and imply that the container can be placed in a centrifugal apparatus shown in FIGURE 2 so that it can be centrifuged around a central axis 1. The container is preferably made of a medical grade plastic material and the polycarbonate material is preferred. Of course, the material should be a light transmitter in the wavelength range of the optical sensor used. The container comprises an outer part of the container 2 and an inner part of the container 3 which are fixed or completely coupled to each other and everywhere remaining in close contact with each other away from the portion where an intermediate channel 4 extending axially is provided. The channel 4 is provided by a notch formed in the internal part of the container 3. The two parts of the container, 2 and 3, comprise their respective lower parts 5 and 6, respectively, said lower parts define a central opening 7 that allows the passage of a piston rod 8. Around the opening 7, the two container parts comprise axially extending portions, 9 and 10, respectively, which extend tightly to the hollow piston rod 8 in a direction away from the interior of container parts. The outer part of the container 2 supported or brought into contact with the hollow piston rod along a small radially extending projection 11, is provided with a recess 12 receiving a sealing ring 13. As illustrated in FIG. 1, the channel 4 continues between the internal and external part of the container the entire route of the external cylindrical walls of said internal and external part of the container along the lower parts 5, 6 and the axial portions 9 and 10 to the opening immediately below the sealing ring 13 in the opening 7. The axial portion 10 of the internal part of the container 3 abutting or coming into contact with the opening 7 is dimensioned so that a narrow but free passage exits inside the parts 2 and 3 of the container around the hollow piston rod 8. The outer part of the container 2 comprises a cylindrical portion of a uniform diameter, compare FIGURE 1. Downward, when viewed relative to the pattern, this portion continues in a portion cylindrical 14 of a slightly large diameter through a short transition portion 15 which forms a frusto-conical internal surface 16. The inner part 3 of the container ends in the location where the transition portion 15 of the outer portion 2 of the container continues in, the cylindrical portion 14 of a large diameter. The lower end of the inner part 3 of the container comprises an external surface 17 of a frusto-conical shape which conforms to the shape of the frusto-conical surface 16 on the inner side of the external part 2 of the container. An inner and an outer annular disc, 19 and 20, respectively, are provided immediately below the lower end of the inner part 3 of the container, which end in a radial surface 18. These discs are brought into close contact with each other, with the exception of the fact, they define between them a channel 21 that extends in an axial plane of a central opening 22 and forward of the internal side of the external part 2 of the container, where the channel 21 communicates with the channel 4 between the outer part 2 of the container and the inner part 3 of the container through an axially extended portion 23. The channel 21 and the axially extended portion 23 are suitably provided by means of a notch on the side of the inner disk 20 which faces the outer disk 19. The two disks 19 and 20 are formed with an oblique course comprising substantially internal and external surfaces, frustoconical, and with which they slope downwards towards the central opening 22 in a direction away from the opening 7 of the hollow piston rod 8 on the external part 2 of the container and the internal part 3 of the container. FIGURE 1 also shows that the inner disk 20 comprises a radial surface 24 that comes into contact with the adjacent radial surface 18 in the inner part 3 of the container. The radial surface 24 of the inner disc 20 is provided with a recess 25 for receiving a sealing ring 26. The two discs 19 and 20 are maintained in the contact position against the radial surface 18 of the internal part 3 of the container by means of a cover 17 for closing the outer part 2 of the container in the downward direction. This cover 17 comprises a circumferential portion in the form of sleeve or sleeve 28 tightly adapted in contact with the inner side of the outer part 2 of the container, to which it is secured in a suitable manner, such as by means of a fast action coupling between a circumferential rod 29 on the outer side of the sleeve 28 and a corresponding circumferential notch 30 on the inner side of the outer part 2 of the container. A sealing connection is secured by means of a sealing ring 31 in a circumferential recess 32 in the outer periphery of the outer disc 19. The cover 27 further comprises a relatively thin wall 32 adapted to form the lower surface of the container in the position shown in FIG. FIGURE 1. This wall 32 extends substantially along a path parallel to the inner and outer disk 19 and 20 so that the wall 32 extends from the inner side of the jacket 27 at a portion adjacent the discs 19 and 20 and descending to a portion substantially on a level with the lower edge 33 of the outer portion of the container 2. To reinforce this relatively thin wall 32, a reinforced radial projection 34 is provided at regular intervals, only one of said projections appearing in FIGURE 1 This projection 34 is formed in part with a portion positioned on the outer side of the wall 32 and partly with a portion positioned on the inner side. of the wall 32, refer to FIGURE 1. The inner back portion is designated with the reference numeral 35 and formed so as to contact the underside of the external disk 19 with the result that aids in the maintenance of the disks 19 and 20 in a reliable position. A separating means 36 tapers between the outer disc 19 and the cover 27. This separation means 36 comprises a length of central tube 37. This. The length or extension of the tube is mounted on a bolt 38 that projects axially inwardly and is integrally formed with the wall 32 of the cover 27. This tube length 37 is formed integrally with a circumferential wall disc 39 which extends outwardly from the length or extension of tube 37 so that initially it tilts slightly downwardly toward the wall 32 of the cover 27 where it then extends along a short axial course to continue in a course which extends substantially parallel to the wall 32 of the cover. The wall disk 39 terminates at a radially extending short periphery 40, which remains on a projection or support 41 at the portions of the rod or rod 35 on the cover 27. An annular filter unit 42 tapers between the outer periphery 40 of the wall disk 39 and the underside of the outer disk 19. This annular filter unit 42 abuts or contacts a surface 43 radially substantially formed on the adjacent outer side of the outer disk 19. To ensure stability in the partition means 36, the radial reinforcing rods or stems designated by reference numeral 44, are additionally accommodated between the length or extension of tube 37 and the wall disc 39. A capsule designated with the general reference numeral 45 is secured at the opposite end of the cover 27 of the tube length 37 of the partition means 36. This capsule comprises a length of the elongated tube 46 formed of integral with a radial disc 47 and carrying two radial and annular discs 48 and 49, additional. These radial discs 48 and 49 are secured by means of an interference coupling on their respective sides of the fixed disc 47. The loose discs 48 and 49 are accommodated at their respective distance from the fixed ring 47 by means of circumferential projections 50 and 51, respectively , in the length of the tube 46. The three discs 47, 48 and 49 are all of the same external 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 is brought into contact with the upper end of the tube length or extension 37 of the partition means 36, thereby determining the position of the capsule 45 in the axial direction. This position is furthermore determined in such a way that when the displaceable sleeve 52 of the capsule travels in the axial direction, a sealing coupling enters at its lower end, contact the drawing, with the innermost edge or edge 53 on the external disc 19 in the central opening 22. In this position of the sleeve 52, there is still a communication between the space inside the internal disk 20 surrounding the sleeve 52 and the entrance opening to the channel 21 between the external disk 19 and the internal disk 20. The axial length of the displaceable sleeve 52 is adapted in such a way that the engagement with the outer disc 20 occurs before the upper end of the sleeve 52, matches the pattern, decouples the fixed ring 47 during the downward axial displacement of said sleeve 52. The internal diameter of the jacket 52 also adapts to the external diameter of the axially extended portion of the wall disc 39 of the partition means 36, such that a displacement Continuous downward movement of the sleeve 52 in the direction of the cover 27 causes the sleeve 52 to be fixedly attached to the partition means 36 once the outer disk 19 is decoupled. The length of the axial portion of the means partition 36 also corresponds to the axial length of the sleeve 52 so that said sleeve 52 in the lowest position is received substantially complete by the partition means 36. As illustrated in the drawing, the hollow piston rod 8 comprises a circumferential piston 55 inside the outer part 2 of the container and the internal part 3 of the container, the piston 55 is sealingly engaged to the inner side of the internal part 3 of the container through a sealing ring 56. A Luer coupling 57 is formed within the hollow piston rod to receive a conventional syringe 58 with a plug 59 which activates the piston to act on the contents of the syringe 58. The coupling 57 is formed in the As a tube extension communicating with a central opening 61 in the piston 55 through a frusto-conical portion 60. The length or extension of the tube 57 is provided with a radial internal projection network 62 to direct the fluid that leaves the syringe 58 away from an axial path and therefore around the length or extension of elongated pipe 46 beneath it within the capsule 45. The last tube extension 46 is of a length or extension and such dimensions can be attached in obturating form the extension of tube 57 within the hollow piston rod 8 when the piston 55 is in its lowermost position near the cover 27. To promote the anterior sealing connection, the inner side of the tube length or extension 57 it is formed with a diameter that gradually decreases at the end adjacent the piston 55. An axial projection skirt 63 is formed integrally with the piston 55 around the ab central section 61 of the piston. This skirt 63 is formed with a diameter and a length that by an adequate displacement of the piston 55, can activate the anterior displacement of the displaceable sleeve 52 of the capsule 45 in the positions in which it engages the internal projection 53 of the opening central 22 through the two discs 19 and 20 followed by a coupling of the partition means 36. An annular rim sealing means 64, resilient, is secured as indicated around the hollow piston in the upper part inside the parts 2 and 3 of the container, make FIGURE 1. This sealing means 64 of the flange is adapted to prevent an undesirable passage of fluid from inside the parts 2 and 3 of the container to channel 4, but this 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 is provided to a hose 65 through of an opening 66 in the external and internal part of the container, 2 and 3, respectively. This connection is known and therefore not shown in greater detail, but this 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 is not shown or described in greater detail. A passage 69 is provided from the area between the partition means 36 and the cover 27 and the entire upward direction through the inside of the length of the tube 37 of the partition means 36 and through the interior of the length or extension of the partition. tube 46 of the capsule 45. This passage 69 allows a transfer of fluid to the syringe 58 of said area, when the length or extension of the back tube 46 is coupled to the length of the tube 57 inside the piston rod 8. Step 66 is provided in the lower portion of the pin or bolt 38 in the cover 27 by said pin or bolt 38 that is formed with an axial, flat surface, said pin being of a substantially circular cross section. As a result, a space is provided between the pin and the adjacent portion of the inner side of the length of the tube 37. An area 67 is provided immediately below the pin 38 where the partition means 36 have a slightly reduced internal diameter. In this way it is possible to place a small filter 68 immediately below said area, compare FIGURE 1, therefore the fluid must pass the filter before it enters the length of the tube 46 of the capsule 45. The described container comprises a first annular chamber 70 defined internally by the hollow piston 8 forming an internal cylindrical wall 71, and externally by an external cylindrical wall 27 formed by the external part 2 of the container and the internal part 3 of the container. When in the position of conventional use, refer to FIGURE 1, the annular chamber 70 is defined upwardly by an upper wall 73 formed by the lower surfaces 5 and 6, respectively, of the outer part 2 of the container and the inner part 3 of the container. container. In descending manner, the annular chamber 70 is defined by a lower wall 74 formed by the piston 55. A second chamber 75 is defined below the piston 55, the second chamber is defined downwardly by the same cylindrical external wall 72 as the first chamber 70. Downwardly, the second chamber 75 is defined by a second lower wall 76 formed by the outer disc 19 and the inner disc 20. The capsule 45 fits centrally into the interior of the second chamber 75. A third chamber 77 is provided below. of the second lower wall 76, and this third chamber 77 is defined by the partition means 36 and the annular filter unit 42. Furthermore, this third chamber 77 communicates with the second chamber 75 through the passage formed by the central opening 22 on the outer disk 19 and the internal disk 20. Finally, a fourth chamber 78 is provided below the partition means 36, the fourth chamber 78 is defined downwardly by the wall 32 of the cover 27 and also by the portions of the jacket 28 of the cover 27 and the underside of the external disk 19.

As described above, the container in question is primarily suitable for the separation of a component, such as a fibrin monomer from the 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, which can catalyze the cleavage of fibrinopeptides A and / or B of fibrinogen, ie convert fibrinogen to fibrin, such as batroxobin. As understood from EP-PS No. 592,242, any enzyme similar to thrombin can be used. Such enzymes include thrombin by itself or any other material with similar activity, such as Ancrod, Acutin, Venyyme, Asperase, Botropase, Crotabase, Flavorxobin, Gabonase, and the preferred Batroxobine. 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 lower 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 is arranged, said syringe containing a buffer of pH-4 prepared from an acetate diluted with acetic acid. The syringe 58 is used later to receive the desired fibrin monomer solution. Another damper known to the person skilled in the art can also be used. The redissolving buffer agent can be any buffered acid solution preferably those having a pH between 1 and 5. Suitable examples include acetic acid, succinic acid, glucuronic 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 acetic acid salts, for example sodium acetate, are preferred. Also, the solubilization can be carried out at a neutral pH by means of a chaotropic agent. Suitable agents include urea, sodium bromide, guanidine hydrochloride, KCNS, potassium iodide and potassium bromide. The concentrations and volumes of such acid buffer or such chaotropic agent are described in EP-PS No. 592,242. During or immediately after the blood supply, the piston rod 8 is pushed so far inside the container that the displaceable sleeve 52 of the capsule 45 moves downwardly within a sealing coupling in the passageway through the vessel. lower wall 76 and second chamber 77. As a result, access is simultaneously opened to the biotin-batroxobin composition within the uppermost chamber 80 of the capsule. When the container is ready for use, a blood sample is fed into the first chamber through a needle not shown and the hose 65 in a conventional manner, said blood sample being preferably mixed with an anticoagulant also of conventional way. During the feeding of the blood through the hose 65 and the opening 66 into the interior of 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 closes in a sealing manner. Subsequently, the container with the blood is placed in a centrifugal apparatus which assists inter alia in the sealing compression of the various portions. The centrifuge apparatus is further described below and causes the container 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 is held radially within the portion remnant of blood, the remnant portion contains the red and white blood cells. As described in EP-PS No. 592,242 the platelets can be present in any fraction, as desired, varying the speed and time of centrifugation. When the interface or contact surface between the plasma and the remaining portion of the blood has stabilized, that is, when the separation is complete, a reduction in the volume of the first chamber 70 is initiated by the piston rod 8 and consequently the piston 55 is ejected. As a result, first a possible inner layer of the air passage passes through the channels 4 and 21 in 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 stops when the entire layer of the plasma has been forced into the second chamber 75, that is, when the contact surface 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 batroxobin of the enzyme with the result that the fibrin monomer, which is polymerized immediately to a non-crosslinked fibrin polymer, is released from the fraction of plasma. This procedure is performed while the container is being centrifuged continuously with the result that the fibrin polymer is efficiently separated from the remaining portion of the plasma fraction, the fibrin polymer is formed by the reaction of the biotin composition. batroxobin and sedimented as a viscous layer along the cylindrical outer wall 72. When this separation has been completed, the centrifugation is stopped, whereby the relatively remaining fluid portion of the plasma fraction can easily be compressed from new in the first chamber 70 by the piston 55 which first rises or rises to transfer air from the first chamber 70 to the second chamber 75 followed by the piston 55 being pressed downwards. This transfer can be carried out relatively easily and quickly before the viscous layer with fibrin polymer reaches the opening to channel 21. Additional measures can optionally be taken to prevent the viscous layer from reaching the entrance of channel 21 too quickly, so that an upshift gear ring 82 is provided shown by dotted lines at the bottom 76.

Once the remaining portion of the plasma fraction has been expelled from the second chamber 75, the displaceable sleeve or sleeve 52 of the capsule 45 is further moved downward so that access to the lowermost chamber 81 is allowed. At the same time or in connection with the last displacement of the sleeve or sleeve, the plug or piston 59 of the syringe 58 is completely pressed downwards by means of a spindle acting from the outside, so that the buffer pH 4 is transferred to the second chamber 75, this may occur while centrifugal stirring is initiated. Addition of the buffer to pH 4 provides that the fibrin polymer dissolves therein, and the presence of the avidin-agarose composition in the lower chamber 81 within the capsule 45 implies that the biotin-batroxobin composition binds a conventional way by avidin. A continuous displacement of the piston 55 causes the displaceable sleeve 52 in the capsule 45 to engage the partition means 36 and disengage the bottom wall 76 resulting in free access being provided for 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 performed during the centrifugal stirring which involves centrifugation and a series of stopping and starting of forward / backward shaking movements. A continuous centrifugation has the effect that the monomeric fibrin solution can be separated in the third chamber through the annular filter unit 42 which retards the relatively large agarose particles and the batroxobin is bonded thereto. When the fibrin monomer solution has passed to the lower fourth chamber 78 as a result of the above centrifugation, said centrifugation is stopped and the fibrin I solution is easily transferred to the syringe 58 by a renewed retraction of the piston 59, the The uppermost end of the length or extension of the tube 46 of the capsule 45 is coupled to the length of the tube 57 that forms the connection with the syringe 58. The described handling of the container shown in FIGURE 1 is carried out in a centrifugal apparatus of the type shown in diagram form in FIGURE 2. The apparatus shown in FIGURE 2 comprises a rotating flat support 101 which is rotatably articulated in a housing not shown in greater detail by means of a ball bearing 102. The flat support Rotary 101 is formed integrally with a vertical motor shaft 103. The motor shaft or actuator shaft is conne through a coupler 104 to a motor 105 which causes that the rotating flat support follows a rotary movement around a vertical axis of rotation. An actuating rod or lever 106 is rotatably articulated coaxially with the axis of rotation within the motor shaft 103 of the rotating flat holder 101, said actuator rod 106 is conne through a coupler 107 with a spindle motor 108 with a needle 109 in such a way that when the spindle motor 108 is activated the activation bar 106 can be moved vertically inwardly or outwardly to engage or uncouple a container 110 placed in the rotating flat holder 101. The container 110 is arranged in the The upper part of the rotating flat support, the container is of the type shown in FIGURE 1. The piston 55 of the container 110 is actuated by means of the rod of the tubular piston 8, confer FIGURE 1, which projects upwards from the upper end of the container 110. The rod of the piston 8 is activated by a clamping means 113, which in turn is activated by means of a spindle motor 115 through a needle 116 and an ac rod. tivadora (not shown) conne to it integrally. The needle or spindle 116 driven by the motor 115 is also activating the piston 59, compare FIGURE 1, of the syringe 58 through said activation bar.

The clamping means 113 are also pivoted in a housing 118 through a bearing. The housing 118 and the spindle motor 115 are secured to a common carrier indicated by dashed lines with the reference numeral 119. This carrier 119 is movably mounted on a rail 120 and causes it to travel vertically therein by means of an engine 121. The engine 121 co-operates through a spherical needle or spindle with a spherical nut 123 secured stationary in the apparatus such that a rotation of the spherical needle or spindle 122 by means of the engine 121 causes a movement of the conveyor 119 and consequently of the fastening means 113 along the slide 20. According to the present inventive apparatus and methods, the supply or supply of the amount of buffer to pH 4 and consequently the activation of syringe 58, quote FIGURE 1, may be in accordance with a buffer amount, fixed, predetermined, to be added or may be performed in response to the amount of non-fibrin polymer crosslinked present in the second chamber 75 of the container 110. According to the present invention, the amount of the fibrin polymer present in the second chamber 75 is measured by means of a photometer 130 arranged in a stationary manner in the apparatus opposite the position of the second chamber 75. The photometer comprises a light emitting device and a light sensor (not shown) arranged so that the intensity of the light can be measured through the wall of the container 110. Any light emitter can be used depending on the material of the wall. The preferred light emitters are in the wavelength range of 400-1100 nanometers. Preferred LEDs that have a wavelength of 920 or 654 mm. The SFH460 Model from Siemens is suitable for this purpose. This photometer 130 measures the amount of fibrin polymer in the outer wall of the second chamber 75 by observing the decrease in light intensity transmitted through the wall of the chamber having the polymer therein when compared to a reference reading of the transmission of light through the wall alone. The transmission readings can be performed continuously for at least the period beginning immediately before the addition of the buffer and ending after the addition has been completed. At the beginning of this period, the thickness and consequently the amount of fibrin polymer are recorded, and the amount of buffer pH 4 to be added is determined based on this. The most recent determination of the buffer amount at pH 4 is made in a control unit 131 that receives the information in the measured values of the photometer through the conduit 132. Subsequently, the control unit 131 activates the motor 115 through a duct 133 so that it drives the spindle 116 and hence the activation bar or lever which in turn activates the piston 59 of the syringe 58. In another preferred embodiment illustrated in Figure 2a, a photometer 130 can be observed. ' As described above, the light transmission of the light emitter is preferably continuous during the deposition process of the polymerized component of the plasma / serum liquid in the wall of the light transmitting chamber. FIGURE 2a shows, in partial cross section, the ratio of the polymerized component, the liquid (plasma or serum) and the piston 55 during the centrifugal deposition of the polymerized component in the wall. Since the liquid can interfere with the accuracy of the data observed by the first photometer or 130, the second photometer 130 'is placed in a position where it is expected that light will be transmitted through the wall and liquid, but not through of the polymerized component. A comparison of these readings can eliminate the possible interference of the liquid in the accuracy of the readings. It is also useful to note that in another preferred embodiment of one or more photometers, 130 and 130 ', should be modulated so that the detector portion remains "activated" but the LED's press "" on and off. "In this way, the detector portion of the photometers can be taken into account (and programmed). to neglect or neglect) the backlight which may be in the vicinity of the present apparatus The modulation of LED's is preferably at a frequency which is not equal to, or is not a multiple of, the rotation speed of the centrifugal apparatus FIGURE 3 illustrates a graph of the measurements of the photometer against time The graph shows the measurements of the photometer from the start of the centrifugal apparatus at the end of the supply of buffer at pH 4 to the second chamber 75. The portion of the graph of A to B shows the measurements taken when the piston 55 is in a lower position and blocks the passage for the photometer signal.In C the piston has been lifted, and the photometer is measuring the transmission. of light through the plastic material only of which the container is made, while the second chamber 75 is still empty. The C-measurement is used to calibrate the photometer so that the measurements that occur take the translucency of the container 110 into consideration, the translucency varies from container to container. From C to D the plasma is transferred to the second chamber 75 together with some air. From D to E the air in the plasma is removed, and the enzyme, such as batroxobin, is released in the second chamber 75. Around the point E, the measurements also provide information of characteristics, so that the concentration and clarity of the blood, which can vary from blood to blood portion. From E to D, the non-crosslinked fibrin polymer is released from the plasma fraction. From F to G, the relatively remaining fluid portion of the plasma fraction is transferred to the first chamber 70 by air which is first drawn from the first chamber 70 to the second chamber 75 by the piston 55 which rises. Then the plasma fraction of remaining fluid is transferred to the first chamber 70 by the piston 55 which is lowered or lowered. During the last period, more centrifuges and piston activations are performed with the result that the entire fraction of fluid plasma is removed from the fibrin polymer. In G the measurements show the thickness of the pure fibrin polymer and hence the amount of fibrin polymer present in the second chamber 75. Based on the last measurement, the amount of buffer at pH 4 added is determined. From G to H, the fibrin polymer solution occurs by means of the buffered buffer at pH 4 supplied.

When the desired amount of buffer at pH 4 has been added, the activation of piston 59 of syringe 58 is stopped. The amount of buffer at pH 4 optionally remaining in syringe 58 is not ejected until later in the second chamber, where it is ejected immediately before the extension of tube 46 is coupled to syringe 58 for suction of the solution of the fibrin monomer from the fourth chamber 78. According to the present invention and as described above the amount of the fibrin polymer deposited on the wall can be determined using a photometer comprising a light source, eg, LED, laser or another light emitter, and sensor arranged to measure the decrease in light transmission through the chamber wall when the fibrin is deposited therein. Any convenient photometer can be employed and the wavelength range of the light source is selected to be sensitive in the range of the material being deposited and taking the material from the wall under consideration. In the case of depositing fibrin polymer in the wall of the chamber, a light emitting diode (LED) of wavelength of the monometer 654 has been found useful. The decrease in the transmission of light takes taking a reference reading (R) of the light transmission through the wall of the chamber prior to the deposition of the fibrin polymer and subsequently measuring the final light transmission (F) after that the deposition of the fibrin polymer is complete. A correlation between the natural log of a comparison of these light transmission intensities and the fibrin mass can be expressed as (i: Fibrin mass = C "In (R / F) where C = a component coefficient, for example, a fibrin coefficient.

The fibrin coefficient (C) can be established experimentally by stopping the procedure and measuring the fibrin mass in a series of functions for observed values of In R / F. Representing graphically where the fibrin mass measured experimentally against the observed light transmission decrease, it is possible to define a fibrin coefficient (the inclination of the dotted line). Subsequently, the measured decreases in the transmission of light expressed in In (R / F), multiplied by the fibrin coefficient, are indicated by the amount in the amount of fibrin polymer formed and known from a predetermined amount of solvent or buffer to being used to solubilize the fibrin polymer provides the concentration of the resulting monomeric fibrin solution. Obviously, the component coefficient could have been restored for different procedures and different blood or plasma components. This concentration can be expressed as (2) Conc = Fibrin mass where Conc is concentration, Vt is the total volume and where V = MassF + VB where VB is the value of absorber or solvent added to solubilize fibrin.

In accordance with the procedure described above, it has been found that some serum and other proteins from the blood or plasma source may become retained in and around the fibrin polymer deposited on the wall. Indeed, the actual fibrin mass can only be a small portion of the deposited fibrin serum mass. This is dependent on the process used, that is, it can vary according to the speed and time (RPM) of centrifugal spin during deposition of the fibrin polymer in the wall. For example, in the centrifugation plasma (obtained from 120 ml of blood) at approximately 9000 RPM for approximately 5-10 minutes in the presence of sufficient amounts of enzyme which convert fibrinogen to fibrin, it has been found that the mass of Real fibrin is only about 5-10% of the fibrin / serum mass that is deposited on the wall. In the case where the serum is present, the concentration can be expressed as (3) Conc = Fibrin mass Vs + VB where Vs = the volume of fibrin and serum retained in fibrin.

It has been experimentally determined that the volume of fibrin + serum (Vs) has a linear relationship with the fibrin mass which can be expressed as Vs a + b 'Fibrin mass where a = volume of serum only b = volume of fibrin per mg of fibrin + serum Both (a) and (b) can be experimentally determined by showing by a diagram the measured fibrin + serum mass versus fibrin mass (a) which is the y-intercept, and (b) that is the inclination of the dotted line. This provides the relationship (5: Conc = Fibrin mass VB + a + b * Fibrin mass By substituting formula (1) for "Fibrin mass" in both examples in formula (5), the following expression is provided (6) Conc = C • In (R / F) VB + a + b • C In (R / F) Accordingly, for a given process wherein C, a and b have been determined experimentally as described above, and wherein the volume of the solubilization buffer (VB) is known, the concentration of a solution of blood component, eg, a The monomeric fibrin solution can be determined by observing the decrease in light transmission through a deposited polymer from which the solution is made and using formula (6) above. In other words, a microprocessor that drives the apparatus in accordance with the present invention, can be programmed with the formula and constants experimentally determined above for a given procedure, so that the signals from the readings of the photometer through the wall of the The chamber before and after the polymerization component is deposited therein, will enable the microprocessor to determine the concentration of the blood component solution that will result from the solubilization of the polymerized component with a known amount of buffer or solvent. Further, in accordance with the present invention, metered or metered distribution means for the buffer or solvent may be provided so that varying amounts of buffer or solvent may be used to solubilize the polymerized blood component in the desired solution. In this way, a solution of a desired concentration of the blood component can be produced regardless of the initial concentration of the component in the blood, determining the amount of fibrin polymer (and serum) deposited and introducing a quantity of buffer in response to this information, which will result in the same concentration from process to process. This is done by re-expressing the formula (6) to solve for the amount of buffer (VB) needed to produce a solution of desired concentration (Conc) as subsequently (7; VB = C • In (R / F) ((1 / Conc) - b) - a Accordingly, the present invention provides apparatus and methods for the production of solutions of blood components, for example solutions of fibrin monomers, at constant desired concentrations even when starting with blood or plasma having varying initial concentrations of fibrinogen, for example 1-10 mg / ml, as can be found in the human population. The invention has been described with reference to a preferred embodiment. Many modifications can be made to the desired photometer, apparatus, container materials, methods and components, without departing from the scope of the invention.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned 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 (17)

1. An apparatus for separating fibrin monomers from blood plasma, the apparatus comprises a container with a reaction chamber for receiving the plasma, wherein the reaction chamber is defined by an external wall and comprises means for supplying or supplying the chamber of reaction with an agent for converting the plasma fibrinogen content to a non-crosslinked fibrin polymer, the apparatus further comprises a device for centrifuging the reaction chamber with the plasma and the agent to a sufficient degree to separate the non-fibrin polymer. crosslinked from the plasma, to deposit the polymer on the external wall of the reaction chamber, and to expel the remaining or residual plasma from the reaction chamber, wherein the container comprises means for administering or supplying to the reaction chamber with a solvent for dissolving the non-crosslinked fibrin polymer, the improvement in ~ -ar? _é "the apparatus comprises a device for measurement for measuring the thickness of the deposit of the non-crosslinked fibrin polymer in the external wall, as well as a control unit for controlling the addition of the solvent in response to the amount of polymer.

2. An apparatus according to claim 1, characterized in that the measuring device is adapted to continuously measure the amount of the non-crosslinked fibrin polymer, at least immediately before and during the addition of the solvent.

3. An apparatus according to claim 1, characterized in that the measuring device is an optical device.

4. An apparatus according to claim 1, characterized in that the measuring device is a photometer.

5. A method for preparing a solution of a blood or plasma component and determining the concentration of the component in the solution, the method is characterized in that it comprises a) subjecting the blood or plasma in a chamber of the apparatus having a light transmitting wall to conditions which catalyze the formation of a polymerized form of the blood or plasma component; b) depositing the polymerized form of the component in the light transmitting wall; c) using a comparison of intensities of a constant light transmission through the light transmitting wall with and without the polymerized component therein to determine the amount of the polymerized component; and d) combining the information obtained in step (c) with the amount of a buffer or solvent used to solubilize the polymerized component in the blood or plasma component solution to arrive at the concentration of the component in the solution.

6. The method according to claim 5, characterized in that the blood or plasma component is the fibrin monomer.

7. The method according to claim 5, characterized in that the polymerized form of the component, polymer mass is determined from the comparison of light transmission intensities using the formula Polymer mass = C * In (R / F) where C = a component coefficient; R is the light transmitting intensity through the wall alone; and F is the intensity of light transmission through the wall plus polymer.

8. The method according to claim 7, characterized in that for a constant process of preparation a component solution provided the coefficient of the component, C, is the slope of a polymer mass measured in the diagram against In (R / F).

9. The method according to claim 5, characterized in that the concentration, Conc, is determined by the formula Conc = C • In (R / F) VB + C In (R / F) where VB = the volume of buffer or solvent to be added.

10. The method according to claim 5, characterized in that some of the plasma serum is retained in and around the polymerized component.

11. The method according to claim 10, characterized in that the concentration, Conc, is determined by the formula Conc = C 'In (R / F) VB + a + b • C In (R / F) wherein VB = the volume of buffer or solvent; a = the volume of serum alone; and b = the volume of per mg of the polymer / serum mass of the polymer.

12. The method according to claim 5, characterized in that the amount of buffer or solvent is controlled in response to the determination of the amount of polymerized component in step (c) to provide a desired concentration of the component solution.

13. The method according to claim 12, characterized in that the amount of buffer or solvent, VB, can be obtained by replacing the desired concentration, Conc, in the formula VB In (R / F) ((1 / Conc) - b)

14. An apparatus for preparing a solution of a blood or plasma component, this apparatus comprises means for determining the concentration of the component, characterized in that it comprises a) means for depositing a polymerized form of the blood or plasma component on a light-transmitting wall of a reaction chamber of the apparatus: b) optical means for determining the amount of the polymerized component deposited on the surface; c) means for solubilizing the polymer with a solvent to provide the solution of the component; and d) means for calculating the concentration of the solution from the amount of polymerized component deposited and the amount of solvent used.

15. The apparatus according to claim 14, characterized in that the component is a fibrin monomer selected from fibrin I, fibrin II or fibrin BB. 16. The apparatus according to claim 14, characterized in that the means for deposit comprises i) a reaction chamber with an external wall, this reaction chamber receives the blood or plasma and which additionally includes a reagent to catalyze the polymerization of the component of the blood or plasma or which also includes means for introducing the reagent into the chamber; and ii) means for rotating the chamber around the longitudinal axis thereof so that when the blood or plasma is rotated in the chamber and subjected to the reagent, the component is deposited as a polymer in the external wall.

16. The apparatus according to claim 14, characterized in that the optical means comprise a photometer which in turn comprises one or more light sources of constant wavelength and sensors for the same, the light sources are arranged in the apparatus of so that the difference in intensity between the transmission of light through the wall of the reaction chamber, alone, and through the wall with the polymerized component therein can be measured.

17. The apparatus according to claim 16, characterized in that the photometer is a signal communication with control means which measure the amount of the solvent used to solubilize the component so that a solution of desired concentration is produced.