Methods for preparing autologous fibrin glue

IS 2 340 705 T3

DESCRIPTION

Methods for preparing autologous fibrin glue.

Cross reference to related requests

This application is a partial continuation and claims priority over United States Application No. 09 / 446,729 filed July 13, 2001 which was issued as United States Patent No. 6,368,298 B1, which is an application of 35 USC § 371 and claims priority over international application No. PCT / IT98 / 00173 filed on June 24, 1998, which claims priority over Italian application No. MI97A001490 filed on June 24, 1997. This application claims priority over each of the applications mentioned above.

Background of the invention

The present invention relates to a method for preparing a solid fibrin network.

Fibrin glue is known to be a blood product that is widely used as a topical surgical adhesive or hemostatic agent. There are several kits available on the market that contain concentrated fibrinogen from donors, associated with a protein activator of human or animal origin, such as thrombin or batroxobin, to obtain heterologous fibrin glue.

Such known kits involve the use of material of human or animal origin which, due to its origin, could lead to possible viral contamination and serious risks to the recipient of the fibrin glue. In the past, the authorities have been forced to withdraw from the market or even ban blood products obtained using material of human or animal origin. Furthermore, cases of rejection caused by reimplantation of fibrin produced using human or animal proteins in patients are known in the literature. Said cases are in fact due to the heterologous origin, with respect to the target organism, of the sealant protein that is reimplanted or some of the components used to prepare it.

Autologous fibrin glue, that is, fibrin glue obtained autologously from a patient's own blood, is more reliable with respect to the risks of rejection and / or infection. Various procedures for making improvised autologous fibrin glue have already been described, but no "ready to use" kit is commercially available although some relevant references can be found in the patent literature.

US Patent No. 5,733,545 describes a plasma buffy coat concentrate for combining with a fibrinogen activator to form a wound sealing platelet glue. The method described in this patent allows a patient's blood to be processed to obtain autologous fibrin glue, but the methods use thrombin or batroxobin as a fibrinogen activator. These activators are of human or animal nature and therefore involve the risk of rejection and / or viral infections for the patient.

WO 98/58689 describes a ready-to-use kit for preparing autologous fibrin glue. It comprises a hermetically sealed container containing calcium chloride as a coagulation activator and possibly tranexamic acid or epsilon-amino-caproic acid as a fibrin stabilizer. To produce autologous fibrin glue, venous blood is drawn from a patient, for example using sterile test tubes. The test tube is then placed in a suitable centrifuge. The sample is centrifuged, thereby separating the red blood cells from the citrated plasma. The test tube containing the separated plasma is kept capped under sterile conditions and placed vertically on a rack to recover the plasma itself. The outer portion of the test tube cap is then sterilized using denatured alcohol and then a sterile needle, which is connected to a sterile syringe, is inserted into the cap of the test tube. The needle is separated 3-4 mm from the separation meniscus of the two phases, and 4 ml of plasma is withdrawn. Using the same needle, the lid of the aforementioned container, which has been previously sterilized using alcohol, is pierced. The citrated plasma contained in the syringe is completely absorbed into the container. This is gently shaken and, after approximately two minutes at 37 ° C, a sterile autologous fibrin tail clot is obtained.

WO 98/11925 describes a system for preparing autologous fibrin glue comprising a sealed container for containing a centrifuge separation medium and a sealed secondary container for containing a calcium clot activator. A connecting tube is provided to connect the primary and secondary vessels.

US 6,083,383 describes a system for preparing an autologous fibrin glue comprising a hermetically sealed primary container to contain a separation medium and a hermetically sealed secondary container to contain a calcium clotting activator.

US 5,030,215 discloses a further system for preparing an autologous fibrin glue comprising a sealed centrifuged primary container and a sealed secondary container containing a calcium clotting activator. The vessels are connected by a conduit.

IS 2 340 705 T3

US Patent No. 5,555,007 describes a method and apparatus for preparing concentrated plasma for use as a tissue sealant. The method consists of separating plasma from whole blood and removing the water from said plasma by contacting it with a concentrator to provide concentrated plasma which can then be coagulated with a solution containing thrombin and calcium. The apparatus comprises a first centrifuge separator in a first chamber, a concentrator (for example dextranomer or polyacrylamide) included in a second chamber communicating with the first chamber, and a second separator. The method described in this reference is time consuming to obtain the plasma concentrate necessary for the subsequent preparation of autologous fibrin glue, and the apparatus is expensive and non-disposable. The method does not describe the use of a calcium clotting activator, and requires a pre-concentration step.

Many methods and systems require the transfer of a fluid from one container to another. For example, many chemical and medical devices require the transfer of a required volume of liquid to react sequentially with various reagents and specific volumetric aliquots. A common practice is to remove the closures from two containers and pipet liquid from one container to the other. This practice, however, exposes the sample to environmental contaminants. For example, this technique is used to transfer plasma that has been separated from red blood cells in a blood sample. A special technique is required, however, to remove the plasma at the interface meniscus. High-density, undesirable lower-fraction red blood cells frequently contaminate aspirated plasma. To avoid this problem, the pipette is frequently kept at a safe distance from the meniscus (ie, the separator between plasma and red blood cells), thereby causing incomplete transfer of the sample. Incomplete transfer of the desirable fraction produces sub-optimal volume yield and non-stoichiometric ratios of the reagents in the sample and those in the second container. This second condition can be a major source of variation in product performance. This is the case for many enzymatic reactions in which reaction rates peak at certain stoichiometric ratios and decline rapidly to higher or lower ratios.

In general, methods and systems are desired for preparing autologous fibrin glue or a solid fibrin that is capable of regenerating tissue in a living organism.

Detailed description of the drawings

Figure 1 is a perspective view of a reference example of a system that does not belong to the claimed invention but illustrates certain features of the invention.

Figure 2 is a cross-sectional view of a main container of the example shown in Figure 1.

Figure 3 is a cross-sectional view of a different embodiment of the main container of Figure 2.

Figure 4 is a cross-sectional view of a different embodiment of the main container of Figure 2.

Figure 5 is an enlarged partial cross-sectional view of a portion of the example of Figure 1 depicting a first end of a transfer device beginning to pierce a sealed main container.

Figure 6 is a view similar to that shown in Figure 5 showing the first end of the transfer device completely piercing the sealed primary container and a second end of the transfer device completely piercing a sealed secondary primary container.

Figure 7 is a view similar to Figure 2 showing the main tube and its contents inverted.

Figure 8 is a top plan view of the example shown in Figure 1.

Figure 9 is a partial cross-sectional view of Figure 8 showing the primary container, the secondary container, and the coupled transfer device, and the contents of the first container being transferred to the second container.

Figure 10 is a top plan view of a kit depicting a reference example of a system that does not belong to the claimed invention but illustrates certain features of the invention.

Figure 11 is a perspective view of an embodiment for carrying out the invention.

Figure 12 is a cross-sectional view of the second embodiment of the invention shown in Figure 11.

Figure 13 is a cross-sectional view similar to Figure 12 showing the reservoir and the main collection device piercing the main collection device.

Figure 14 is a cross-sectional view similar to Figure 12 showing the reservoir piercing the main collection device, and emptying its contents into the device.

IS 2 340 705 T3

Figure 15 is a perspective view of a further embodiment for carrying out the invention.

Figure 16 is a cross-sectional view of a third embodiment of the invention shown in Figure 15.

Figure 17 is a perspective view of a transfer device depicting a reference example not belonging to the claimed invention but illustrating certain features of the invention.

Figure 18 is a cross-sectional view taken along line 18-18 in Figure 17.

Before embodiments of the invention and reference examples are explained in detail, it should be understood that the invention is not limited in its application to details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways. Furthermore, it should be understood that the phraseology and terminology used in this document are for the purpose of description and should not be construed as limiting.

Summary of the invention

In one aspect, a non-inventive reference example provides a system for preparing an autologous solid fibrin network suitable for regenerating tissue in a living organism. The system comprises a hermetically sealed main container containing a separation medium and a low density, high viscosity liquid. The separation medium is capable of separating red blood cells from plasma when the container contains blood and is centrifuged, and the main container has a first pressure. The system further comprises a hermetically sealed secondary container containing a calcium clot activator. The secondary container has a second pressure that is less than the first pressure. The system also comprises a transfer device that includes a cannula having a first end and a second end. The first and second ends are capable of piercing the sealed primary and secondary containers to provide fluid communication between the first and second containers. The low-density, high-viscosity liquid in the main container is able to block flow through the cannula after entering the cannula.

In another aspect, a non-inventive reference example provides another system for preparing a solid fibrin network capable of regenerating tissue in a living organism. The system comprises a hermetically sealed main container that has a first pressure that is capable of drawing blood from its interior. The system further comprises a hermetically sealed secondary container having a second pressure and containing a calcium coagulation activator. The second pressure is less than the first pressure. The system also comprises a transfer device that includes a cannula having first and second ends. The first and second ends are capable of piercing the sealed containers, and the transfer device is capable of transferring a part of the blood drawn in the main container to the second container by pressure difference. The system also includes a centrifuge to simultaneously centrifuge and coagulate the portion of blood transferred from the primary container to the secondary container through the transfer device and brought into contact with the calcium clotting activator to form a solid fibrin network. which is capable of regenerating tissue in a living organism.

In another aspect, a non-inventive reference example provides a method of preparing a solid fibrin network for regenerating body tissue in a living organism. The method comprises drawing blood from a patient in a main container and separating the plasma from the blood in the main container. Plasma from the primary container is transferred to a secondary container containing a calcium clot activator using a transfer device comprising a cannula having a first end and a second end for contacting the plasma with the clot activator. of calcium. Plasma and calcium clot activator are coagulated and centrifuged simultaneously in the secondary vessel to form a solid fibrin network. The solid fibrin network is suitable for regenerating body tissue in a living organism.

In another aspect, a non-inventive reference example provides another system for preparing a solid fibrin network capable of regenerating tissue in a living organism. The system comprises a hermetically sealed main collection device having an interior and containing a separation means. The main collection device is capable of containing drawn blood inside, and the separation means is capable of separating plasma from red blood cells when the main collection device contains blood and is centrifuged. The system further comprises a reservoir having a chamber and a conduit in fluid communication with it. The chamber has a calcium coagulation activator within it, and the conduit is at least partially filled with a blocking means to prevent the activator from flowing from the chamber under ambient conditions.

The invention defined by claim 1 provides a method for preparing a solid fibrin network capable of regenerating tissue in a living organism. The method comprises drawing blood from a patient in a main collection device having a seal and providing a reservoir that includes a chamber and a conduit in fluid communication with the chamber. The chamber is at least partially filled with a calcium coagulation activator, and the conduit is at least partially filled with a blocking means to prevent the activator from flowing from the chamber at ambient conditions. The reservoir is connected to the main collection device so that the chamber, conduit, and collection device would be in fluid communication were it not for the

ES 2 340 705 T3 locking means. The main collection device is then centrifuged at a first speed. The first speed is sufficient to separate the plasma from the blood, but not sufficient to move the blocking means in the conduit to the main collection device. The main collection device is then centrifuged at a second speed. The second speed is sufficient to move at least a part of the blocking means from the conduit to the main collection device, thus allowing the calcium coagulation activator to flow into the collection device and come into contact with the plasma, forming thus a solid fibrin network suitable for regenerating tissue in a living organism.

Detailed description of the invention

This application is a partial continuation and claims priority over United States Application No. 09 / 446,729 filed July 13, 2001 which was issued as United States Patent No. 6,368,298 B1, which is an application of 35 USC § 371 and claims priority over international application No. PCT / IT98 / 00173 filed on June 24, 1998, which claims priority over Italian application No. MI97A001490 filed on June 24, 1997.

The object of the present invention is, therefore, to provide a ready-to-use kit that allows to quickly obtain autologous fibrin glue and does not cause viral infections and / or cases of rejection when used in surgery.

Said object is achieved by using a coagulation activator, which is not of human or animal origin, but an inorganic compound that therefore cannot be infected and cannot cause rejection. In particular, the object is achieved by a method according to the invention 1.

The "ready to use" kit according to a reference example which is not the present invention comprises a hermetically sealed container containing calcium chloride as a coagulation activator. Calcium chloride activates fibrinogen present in a patient's plasma when it is introduced into the hermetically sealed container.

The systems and kits according to the present examples for carrying out the invention have the great advantage of allowing the preparation of autologous fibrin glue that can be used without risk of viral infections or cases of rejection. Another advantage of the kit according to the reference example is that it allows the preparation of autologous fibrin glue from the patient's plasma in a very short time as well as in the formation of clots or membrane or spraying. Yet another advantage of the kit prepared for use according to the reference example is that it allows the autologous fibrin glue to be obtained at proportionally lower costs with respect to known systems.

Additional advantages of the kit according to the present invention will be apparent to those skilled in the art from the following detailed description of some embodiments thereof.

Suitable containers for the kit according to the present invention include a glass container for antibiotics as described hereinafter in Example 1. Glass or plastic test tubes can also be used. The preferred volume of the container is from 5 to 15 ml. The test tubes preferably have a diameter ranging from 12 to 16 mm and a height ranging from 75 to 100 mm. The container must be adequately thick to withstand the stresses caused by the pressure difference between its internal space and the atmosphere when evacuated. The hemispherical or conical bottom tubes are preferably 0.7 mm thick, the flat bottom tubes are 1 mm thick. The plastic containers are preferably made of transparent polyester resin, 0.2-0.8 mm thick, to ensure maintenance of vacuum for at least 12 months after production. After preparation, the plastic test tubes are preferably placed in a tinplate vacuum sealed container having an inner layer of heat-sealed polyethylene to ensure a perfect seal until the date of use.

It should be noted that evacuation of test containers or tubes is advisable, although not necessary to practice the present invention.

The containers or test tubes are hermetically sealed by means of pierceable rubber or silicon stoppers, which are suitable to ensure that the container is perfectly hermetic and to allow vacuum closure after the introduction of the chemical components and before the step of steam or radiation sterilization.

After sealing, the containers can be steam sterilized at 121 ° C for 30 minutes. Sterilization can also be done by gamma ray or electron beam irradiation.

Although a fibrin stabilizing tranexamic acid can be used, pure crystalline epsilon-aminocaproic acid is also suitable. The amount will be about 1 g when using a 25 ml container, suitable for a 20 ml amount of plasma. Sometimes it is not necessary to use a fibrin stabilizer.

CaCl is used as a coagulation activator₂-2H₂Either or a liquid solution containing calcium in the present invention although different coagulation activators (listed below) can be used. For example, 11.76 mg of CaCl will be entered₂-2H₂Or in a 5 ml container, using a precision dosimeter (maximum error: 1-2 mg), to avoid introducing contaminating foreign components.

IS 2 340 705 T3

In the case of a 15 ml container for a 12 ml amount of plasma, the amount of solid dehydrated calcium chloride to be introduced will be as high as 35.28 mg, while the amount of tranexamic acid will be proportionally as high as 300 mg. of crystals.

In the case of a 25 ml container for a 20 ml amount of plasma, the amount of dehydrated calcium chloride to be introduced will be as high as 58.8 mg while the amount of tranexamic acid will be proportionally as high as 500 mg of crystals. .

In addition, from the dehydrated form used in the Examples, the calcium chloride may be in any other suitable form available on the market, for example in the form of CaCl.₂-2H₂O. A solution of this salt can also be used, as described in Example 1.

Examples

Example 1 (Reference)

100 mg of tranexamic acid, which acts as a fibrin stabilizer, was placed in a 5 ml glass container for antibiotics, which can be vacuum sealed, made of clear, inert white glass and 1 mm thick. Synthetic tranexamic acid, which is more than 98% pure, is marketed by the American company Sigma Inc. Separately, a CaCl solution was prepared₂ 1 M weighing 147.0 g CaCl on a precision balance₂-2H₂Or (> 99% pure), from the same American company Sigma Inc.

This salt was dissolved in exactly 1 liter of ultra-pure pyrogen-free distilled water, for several minutes at room temperature, with frequent stirring. Using a precision piston dispenser, which has a dispensing accuracy of ± 5% (Eppendorf type), 80 µl of the activator solution was introduced into the glass container. In this stage, at the same time as dosing, a filtering was carried out using a 0.22 jum Millpore sterilizing filter, carefully avoiding the possible contamination of powders or filaments of any kind. Finally the glass container was covered with a pierceable and vacuum sealable rubber lid, taking care not to completely cover the container, to allow subsequent vacuum closure and possibly further sterilization using gas. The container was then placed in a suitable device for vacuum closure, avoiding any possible contamination of solid particles in the atmosphere (ULPA or HEPA filtration in a sterile chamber). A vacuum of up to 4 ml was applied, using a membrane vacuum pump and micrometer control, to the internal atmosphere of the device. To control the level of vacuum in the internal atmosphere, a precision vacuum gauge (precision 1 mbar) was used. Finally, without unloading the device, the container was closed under vacuum, to be recovered later for use as described in the following Example.

Example 2 (Reference)

10 ml of venous blood was withdrawn from a patient according to the conditions of the qualitative standards for clinical analysis, for example using VACUTAINER® sterile test tubes from Becton-Dickinson, to which a 0.106 M sodium citrate solution had been added. Test tubes to which disodium or dipotassium ethylenediaminetetraacetate had been added can also be used for this purpose. The sample was carefully kept sterile during blood collection. Finally, the sample was gently shaken to completely mix the components, thus ensuring the anticoagulant action of the sodium citrate. The test tube was then placed in a suitable centrifuge, carefully balancing the weight of the rotor to prevent damage to the centrifuge itself. After the lid was sealed, the sample was centrifuged at 3,500 rpm for 15 minutes, thereby separating the red blood cells (which are thicker) from the citrated plasma (supernatant). In this case, the plasma yield, which depends mainly on the characteristics of the donor blood, was up to 55%. The test tube containing the separated plasma was kept closed under sterile conditions and was placed vertically on a shelf to recover the plasma itself, at this stage care was taken not to shake the test tube, to avoid mixing the two phases separated in centrifugation. The outer portion of the test tube cap was then sterilized using denatured alcohol and then a sterile needle, which was connected to a sterile syringe, was inserted into the cap of the test tube. The needle was brought up to 3-4 mm apart from the meniscus of separation of the two phases, and 4 ml of plasma was withdrawn. Using the same needle, the cap of the container according to the present invention, which had been prepared as described in Example 1, was pierced, which had previously been sterilized using alcohol. As soon as the needle pierced the cap, the citrated plasma contained in the syringe was fully drawn into the container. This was gently shaken and after approximately 2 minutes at 37 ° C, a sterile autologous fibrin tail clot was obtained, ready for immediate use.

IS 2 340 705 T3

Example 3 (Reference)

Approximately 18 ml of venous blood was drawn from a 49-year-old normotype patient using VACUTAINER test tubes.^® of Becton Dickinson with 5 ml of sodium citrate, taking care to shake gently immediately after sample extraction. Blood taken in this way was immediately subjected to centrifugation (15 minutes at 2500 rpm) to separate the plasma. Plasma (12 ml) was carefully transferred to two 10 ml test tubes, containing 120 µl of CaCl.₂ (10 g / 100 ml) each, which had been prepared as described in Example 1, but without using tranexamic acid. After mixing the plasma with the activator, the test tubes were centrifuged for 30 minutes at 3000 rpm, finally obtaining two massive fibrin samples that were inserted, with all the precautions of sterility, in 2-3 hours from the preparation, in the large vesicular mandibular cavity resulting from the extraction of the embedded left canine and the second right incisor, as well as the abscission of the cyst present in the central area of ​​the incisor teeth. Finally the edges of the gingiva were closed with eight stitches. An X-ray control 15 days later showed the fibrin still in position, apparently intact. Histology 7 months later demonstrated the complete replacement of fibrin with bone tissue, with better post-operative development than with traditional methods, which required more than 12 months to achieve the same result. Since no antifibrinolytic agent has been used for the preparation of autologous fibrin, it can be stated in this case that said additive was useful for the specific purpose.

Example 4 (Reference)

To produce an adhesive fibrin glue, 12 ml of plasma, obtained as in Example 3, with all measures to preserve sterility, were transferred into a 20 ml container according to the present invention, prepared as described in Example 1.

After carefully shaking, the mixed plasma was poured onto a sterile glass slide, of the type used in chemical laboratories, where the plasma was mixed with sterile and very pure calcium carbonate of colarine origin (BIOCORAL ™ · NOTEBS, SA France), or with calcium fluoride (> 98% Sigma Inc.). These calcium salts are both well known to those skilled in the art as fibroblast stimulators.

By mixing a part of the plasma with a part of calcium carbonate, (for example; 2 ml with 500 mg) a malleable, sterile and adhesive paste was obtained and was used as a filler for the subgingival spaces or different cavities after abscission of infected mucosal sacs. The paste, positioned so as to fill the void spaces, formed in a few minutes a solid fibrin network that acted as a hemostatic plug and created an anthologous biological substrate that supported the mucosal borders in position and where the subsequent migration of connective cells began.

Example 5 (Reference)

To obtain a fibrin glue membrane, 20 ml of plasma, obtained as in Example 3, was placed in a 25 ml flat bottom container according to the present invention prepared as in Example 1. After normal careful shaking, The container was centrifuged for 40 minutes at 4000 rpm with an external spinning rotor. At the end of the centrifugation operation, from the bottom of the test tube, a white colored membrane, very compact and of high tension, was recovered, which was the same size as the bottom of the test tube (24 mm in diameter) and a 3mm thickness. This autologous membrane, due to its compaction and strength, was used as a maintenance and separation membrane in dental and general surgery, as a substitute for porous synthetic membranes. The membrane obtained can be stored sterile for several days at 4 ° C.

Example 6 (Reference)

To obtain large fibrin glue membranes, approximately 200 ml of citrated plasma was withdrawn from a patient, collected, and separated into a double transfusion bag. The plasma was subjected to cryoprecipitation by freezing it at -80 ° C for 12 hours, defrosting overnight at 4 ° C (this procedure is well known to those skilled in the art). The same morning the plasma obtained by this procedure was subjected to centrifugation for 15 minutes at 5000 rpm at 4 ° C to obtain approximately 20 ml of cryoprecipitate. After careful removal of the supernatant using a pressure device (for example XP1000 from the company Jouan SA France) the cryoprecipitate was taken with 20 ml of whole plasma from the same patient. The resulting 40 ml were placed in a sterile 35 mm diameter polypropylene container, bottom

Flat ES 2 340 705 T3, according to the present invention, containing the appropriate amount of activator, as in Example

1. After careful shaking, the container was centrifuged for 40 minutes at 5000 rpm to obtain a membrane as in Example 5, but more compact and stronger in tension due to the higher fibrin content.

Such a membrane can also be stored sterile for several days at 4 ° C.

The membrane obtained by the method described in Example 5, in addition to the use described in Example 4, can be used as a substrate for the in vitro culture of dermal cells from the same patient, to obtain grafts to be transplanted in the event of very serious burns.

Good quality membranes useful for the aforementioned purposes can also be obtained from separated whole plasma transferred directly to the container in accordance with the present invention. The membrane obtained will be thinner than that described above, but it will still be useful for surgical uses and as a substrate for cell growth.

Example 7 (Reference)

To obtain pulverized fibrin from a cryoprecipitate as in Example 5, 20 ml of cryoprecipitate were taken with 10 ml of complete plasma at room temperature and stirred carefully until dissolution was complete. The resulting plasma was carefully transferred to a 50 ml container according to the present invention, prepared as in Example 1, shaking carefully for a perfect mixing of the components. After 120 seconds at room temperature, the test tube was connected to a sterile Venturi-type air compressor, known to those skilled in the art, to distribute it evenly over the surface of a bleeding organ undergoing surgery (lung , heart, spleen, arterial anastomosis). The concentrated plasma, which contained concentrated fibrinogen, thrombin, calcium ions and other coagulation enzymes, was distributed over the organ, coagulated in a few seconds, also due to the tissue coagulation activating enzymes present in the patient's endothelium creating a film of fibrin that had a protective hemostatic function.

The surgical operation therefore concluded with the reduction of internal bleeding, thus avoiding blood transfusions or additional complications.

The present invention also provides a method of forming a solid fibrin network or an autologous tail capable of regenerating tissue in a living organism. In this method, anticoagulated plasma is obtained by centrifugation of a blood sample. The transfer devices described in this document and not forming a part of the invention allow the plasma to be transferred to a second container containing calcium clotting agents and then immediately centrifuged to obtain a dense, autologous network of fibrin and platelets. stable. The transfer devices described in this document can also be used to transfer other liquids in other applications. In other words, the transfer devices and systems described herein enable simultaneous centrifugation and coagulation. Using these systems and methods, several advantages can be achieved: 1) the sample is handled in a manner whereby sterility is maintained; 2) the total volume of plasma is transferred to maximize a complete yield of a clot; 3) the stoichiometric ratio of anticoagulant and calcium clotting agent is kept in a narrow range to minimize the clotting period; 4) the transfer is completed quickly; 5) Health assistants who do not normally perform these operations (eg dentists) can easily perform these methods and operate the systems; and 6) the devices are for single use only to avoid reuse and possible contamination by bloodborne pathogens.

The invention provides a method of preparing a solid fibrin autologous network or tail that can be used to generate tissue in a living organism. In an example that does not pertain to the invention but is useful for understanding the invention (shown in Figure 1), the system comprises a primary container 10, a secondary container 14, and a transfer device 18. Preferably, the main and secondary containers 10, 14 are tubes, and more particularly, test tubes, although any container that is capable of housing a fluid or liquid and being centrifuged is suitable for use with the invention. Preferably, the containers 10, 14 are made of glass or plastic.

The main container 10 must be capable of drawing blood therein using conventional venipuncture techniques. Preferably, the main container 10 is sealed with a seal 22 while the blood is drawn to avoid contamination, although the container 10 may be sealed immediately thereafter. A variety of seals 22 can be used to seal the main container 10, for example, a rubber tampon, lid, foam, elastomer, or other compound. The seal 22 must be capable of being pierced or punctured, and therefore rubber and silicone are preferred materials from which the seal is made, although any material that provides a seal and can be pierced can be used. The main container 10 may contain an anticoagulant solution 25. The anticoagulant 25 in the solution preferably comprises a calcium binding agent. More particularly, the anticoagulant 25 may comprise sodium citrate, disodium salt of ethylenediaminetetraacetic acid, dipotassium and tripotassium salt of ethylenediaminetetraacetic acid, and combinations thereof. Preferably, main container 10 contains a sodium citrate solution. The anticoagulant 25 tends to dilute the blood collected in the main container 10 to make it ready for centrifugation. In addition, the main container includes a means of

ES 2 340 705 T3 density gradient separation 26, air 27 as well as a high viscosity low density fluid 28 (see Figure 10 showing a kit further described below).

The density gradient separation means 26 must be capable of separating different fractions of a particular liquid or fluid in the main vessel 10 having different densities. The separation medium 26 allows the dense and unwanted fractions of the liquid to be separated by centrifugation, and subsequently removed. For example, separation medium 26 can separate red blood cells 30 from platelet-rich plasma 34 during centrifugation of a blood sample. In one example, the separation means 26 may be at the bottom of the main container 10. In other examples, the separation means 26 can be applied as a ring around the interior of the main container 10, or any other suitable interior position. Although any density gradient separation medium 26 capable of separating liquids having different densities during centrifugation is suitable for use with the invention, preferably medium 26 is a gel, and more preferably, a thixotropic gel. Figure 2 illustrates the main container 10 after centrifugation of a blood sample has taken place, and also shows the gel separation medium 26. Preferably, the thixotropic gel has a sufficient creep limit so that it does not flow into or move around the main container 10 under typical ambient conditions, but does flow at higher centrifugal forces than are experienced during centrifugation. More preferably, a gel having a density that is less than the high density of the unwanted red blood cell fraction 30, but greater than the density of the desired plasma fraction 34 is preferred. In other words, most preferred is a gel or other medium that is capable of separating red blood cells 30 from plasma 34 after a blood sample has been centrifuged. Said medium 26 will move or flow within the container during centrifugation, but will not flow thereafter, thereby creating a semi-permanent barrier between the separated fractions when centrifugation is complete.

As shown in Figure 3, another suitable density gradient separation medium 26 that may be employed in the main vessel 10 is a plurality of plastic beads 26 having the desired density for fraction separation. The beads can be suspended in the high-viscosity, low-density fluid required for subsequent sealing of the transfer device 38. During centrifugation, the beads 26 migrate to the interface between the two fractions 30, 34 and are compacted, as by sintering, to form a stable barrier between the fractions having different densities (i.e. red blood cells 30 and plasma 34 ). The low-density, high-viscosity residual fluid that covers the sediments contributes to the stability of the compacted layer.

Other suitable density gradient separation means include polymeric floats such as those described in US Patent Nos. 5,560,830 and 5,736,033 issued to Coleman. Figure 4 shows a polymeric floating device 26.

The high viscosity, low density immiscible fluid 28 ("LDHV fluid") in the main container generally comprises an inert oil. More preferably, the LDHV fluid is polyester, silicone, or other inert fluid, and is applied to the main container at a position above the gel by displacement or pressure pumps. The LDHV fluid must be capable of blocking or eliminating flow through cannula 38 of transfer device 18 after entry into it as further described below.

Secondary vessel 14 (shown, inter alia, in Figures 1 and 10) contains the chemical reagents necessary for the particular reactions. The second container 14 is sealed by a seal 24 in a similar manner as the first container 10, that is, by a rubber stopper, lid, foam, elastomer, or other compound. In one application of the invention as discussed below, the secondary tube may contain a calcium clotting activator 36. Examples of suitable calcium clotting activators include, but are not limited to, calcium chloride, calcium fluoride, calcium carbonate, and combinations thereof, however any calcium-containing salt will suffice as a calcium clotting activator. In addition, other activators include calcium gluconate, calcium fumarate, calcium pyruvate, and other organic calcium salts that are soluble in water and compatible with human life. The clotting activator coagulates plasma when it comes into contact with it. The secondary container 14 can be completely evacuated to an internal pressure that is substantially zero. The evacuation of the secondary container 14 facilitates the transfer of fluid from the main container 10 to the secondary container 14 through the transfer device 18. Since there are no gaseous molecules present when the secondary container 14 is filled during the transfer, there is no compression of the waste gas with the resulting increase in temperature. As a result, flow rate is maximized, complete transfer is facilitated, sterility is maintained by eliminating the need for ventilation, and the desired stoichiometric ratio for the desired reaction is maintained.

In another example, the secondary container may also contain one or more of an antibiotic, an analgesic, a cancer therapeutic, a platelet growth factor, and a bone morphogenic protein. Other therapeutic agents that can be administered topically may also be included. Examples of antibiotics include, but are not limited to, ampicillin, erythromycin, and tobramycin. Pain relievers include, but are not limited to, aspirin and codeine. Cancer therapeutic agents include, but are not limited to, 5-fluorouracil.

The transfer device 18 may comprise two pieces as shown, for example in Figure 1, or alternatively, it may be in one piece as shown for example in Figures 17-18. A single piece, single mold transfer device 18 is preferred. As best shown in Figures 5-6 and 17-18, the transfer device 18 comprises a cannula 38 having a first end 42 having a first

ES 2 340 705 T3 opening 46 and a second end 50 having a second opening 54. The ends 42, 50 of the cannula 38 are sharp or pointed (or even have a beveled surface on them) to be able to pierce or penetrate into seals 22, 24 of primary and secondary containers 10, 14. Cannula 38 is retracted and coaxially mounted within housing 58 to prevent accidental finger pricks during container handling. Housing 58 has two opposing cylindrical guides 62, 64 that are centrally and axially oriented with cannula 38. Guides 62, 64 serve to guide primary and secondary vessels 10, 14 at first and second ends 42, 50 of the transfer device 18. Figures 5 and 6 show guides 62, 64 that guide containers 10, 14 at the first and second ends 42, 50.

The ends 42, 50 of the cannula 38 may be encompassed or covered by safety valves, protective layers, or elastomeric sleeves 68, 72, which form an airtight seal. The safety shields 68, 72 also cover the first and second openings 46, 54. When the first and second ends 42, 50 pierce the elastomeric sleeves 68, 72, the sleeves 68, 72 retract accordingly. Figure 5 shows the first end 42 beginning to pierce the seal 22 of the main container 10 and the sleeve 68 retracting accordingly, while the sleeve 72 still completely covers the second end 50. The ends 42, 50 are extended enough to pierce seals 22, 24 completely, but they do not extend much further into containers 10, 14 (as shown in Figure 6). This allows the transfer of the maximum volume of the liquid volume from the inverted main container 10 to the secondary container 14. Figure 6 also shows the first and second ends 42, 50 that have completely pierced the seals 22, 24 of the first and second containers 10 , 14, and the two covers 68, 72 fully retracted. Elastomeric sleeves 68, 72 prevent gas or liquid flow when unperforated. Suitable materials for sleeves 68, 72 include, but are not limited to, varieties of rubber and thermoplastic elastomers.

Returning now to the operation of the first embodiment, once blood has been drawn into the main container 10 using conventional venipuncture techniques, the blood is treated with anticoagulant by the anticoagulant 25 therein. Typically, the main container 10 is sealed while the blood is being drawn, however, it can be sealed thereafter. The sealing of the main container 10 prevents contamination of the contents inside. Thereafter, the main container 10 and its contents (ie, blood, anticoagulant 25, separation medium 26, and LDHV fluid 28) are centrifuged. Acceptable centrifugation can take place at a gravitational force in the range of 900 to 3,500 xG for 5 to 15 minutes. In a preferred embodiment, the main vessel is centrifuged at a gravitational force of approximately 1000 xG for approximately ten minutes. This initial centrifugation separates the contents or fractions of the main container into a plurality of layers as shown, for example, in Figure 2. The layers include (in order from the bottom of the main container 10 to the top of the container after centrifugation): the red blood cell layer 30, the separation medium 26, the platelet-rich plasma layer 34, the LDHV fluid 28, and finally a volume of residual gas 27 at a pressure equal to atmospheric. The proportions of these layers may vary from application to application, and are shown herein in these proportions for illustrative purposes only. After centrifugation, the sealed main support 10 is inverted before the transfer device 18 is used to pierce the seal 22. In other words, the main container 10 is inverted so that the sealed opening is in the lower vertical position as shown in Figure 7. The inversion of the main container changes the order in which the layers are arranged. Above seal 22 are the following layers in sequence from bottom to top: platelet-rich plasma 34, low-density, high-viscosity immiscible fluid 28, waste gas 27, separation medium 26, and globules red 30.

The secondary container 14 is then placed in an upright position with its sealed opening 24 in the uppermost position as best shown in Figure 8. This positions the secondary container 14 for transfer of the contents of the main support to its interior. Figure 8 illustrates the main container 10 spun in an inverted position over the transfer device 18, which is above the secondary container 14 in the proper position for the transfer. The transfer device guide 64 is then positioned and guides the secondary container 14 therein, while the inverted main container 10 is then placed in the other guide 62 (or vice versa). In other words, either end 42, 50 of cannula 38 can be used to pierce any seal 22, 24. Since transfer device 18 is symmetrical at both ends, the user is provided with a foolproof degree of operation. The user then pushes the containers together to pierce the two seals 22, 24 with each respective end of the cannula 42, 50. The two valve covers 68, 72 covering the ends 42, 50 further enhance foolproof operation. First, if the first end 42 pierces the main seal 22 (again, either end can be used to pierce any seal), the non-perforated sleeve 72 covering the other end 50 will contain the fluid, thereby preventing the fluid from spill. On the other hand, if the other end 50 pierces the other seal 24 (and the sleeve 72 accordingly) first, the vacuum is maintained by the sleeve 68 covering the first end 42.

Once the ends 42, 50 pierce the two sleeves 68, 72 and the seals 22, 24 as shown in Figures 6 and 9, the desired fluid is transferred from the main container 10 to the secondary container 14 by pressure difference. In other words, as the pressure in the secondary container 14 has been released, the contents (more particularly, the plasma 34) of the main container 10 flow into the secondary container 14. The pressure in the main vessel 10, originally at atmospheric pressure, decreases as the level of the liquid decreases and the volume of the gas expands. However, at no time is the pressure equal to zero. Since the secondary container 14 is completely evacuated to a pressure equal to zero, the pressure inside it does not increase as the tube fills since there is no gas to compress. Accordingly, apparatus 18 can be used to transfer

ES 2 340 705 T3 a wide variety of fluids and solutions from tube to tube, and should not be construed as limited to blood transfer only.

Due to the particular sequential arrangement of the layers in main container 10, platelet rich plasma 34 is easily transferred. Furthermore, since the main container 10 is also pre-set to an evacuation level, the container is only partially filled after blood collection. This allows the gas in the "headspace" to remain significantly above zero during the transfer as its volume expands, thereby allowing a complete and rapid transfer to the secondary vessel 14. This is dictated by the gas law. ideals and the Poiseuille-Hagen equation.

Transfer of the contents or fragments of the main container (ie platelet rich plasma) continues until LDHV fluid 28 enters cannula 38. The high viscosity of LDHV fluid plugs the narrow lumen of cannula 38, thereby causing this flow interruption mode. This avoids reuse of the transfer device 18, which is particularly important in attempting to remove blood-contaminated transfer devices, and also prevents accidental contamination by blood-borne pathogens from previous use or by another patient.

Transfer of plasma fraction 34 to secondary container 14 is complete, thus allowing maximum throughput and maintenance of the proper stoichiometric ratio of reagents. Plasma 34 then comes into contact with clot activator 36 in second container 14, thereby creating a mixture 60 that can be immediately centrifuged to form a solid fibrin network. The pressure difference between the main and secondary containers 10, 14 is maintained substantially throughout the transfer, allowing a rapid transfer. The transfer device 18 is not affected by the order of arrangement of the tubes, which makes the system practically foolproof. Finally, the transfer occurs without ventilation, maintaining sterility and the absence of contamination of the sample.

In general, the transfer device 18 provides a fast and efficient way of contacting the plasma 34 with the calcium clotting activator 36, immediately after which simultaneous coagulation and centrifugation of the plasma can take place to form the network. solid fibrin. The solid fibrin network is suitable for regenerating body tissue in a living organism. Such a method alleviates the need to first pre-concentrate the plasma by removing the water from the plasma before the plasma is contacted with the calcium clotting activator 36. In addition, the transfer device 18 can be used to transfer blood. or other fluids in a wide variety of applications.

The invention also provides a ready-to-use kit as shown in Figure 10. The kit comprises a primary container 10, the secondary container 14, and the transfer device 18. In one embodiment of the kit, the kit may have two trays 70, 74 that are taken out of a container. The first tray 70 has all the components required for Stage 1 and the second tray 74 has all the components required for Stage 2. Of course, the components can be arranged in a wide variety of ways.

Step 1 comprises collecting blood in main container 10, followed by centrifugation to obtain platelet rich plasma. The components of the first tray 70 comprise an alcohol swab 78 to clean the venipuncture site, a multiple blood sample collection needle 82 (21 x 1 "gauge), a safety holder 86, the main container 10 containing the anticoagulant (eg, citrate), gel, LDHV fluid, and a bandage 90 to cover the venipuncture site. The venipuncture site is cleaned with the sterile alcohol swab 78. The needle cartridge 84 is opened and screwed into the safety holder 86. The needle 82 is then inserted into the patient's vein and the container 10 is connected to the holder 86. The blood then fills the container, and the needle 82 is removed and inserted back into bracket 86. The end of the bracket is closed with the flap folded. The vein is closed with bandage 90. Vessel 10 is centrifuged at approximately 1000 xG for approximately 10 minutes and the plasma is separated from the red blood cells.

The second tray components are the components used for stage 2 and include an AFT (Autologous Fibrin Tube) tube or secondary container 14 and a transfer device 18. Stage 2 comprises placing the main container 10 in an inverted position and in transfer device 18. Secondary container 14 contains the coagulator and is pierced through the other end of the transfer device. The vessels 10, 14 are pooled and the platelet rich plasma flows from the main vessel 10 to the secondary vessel 14. The secondary vessel is then immediately centrifuged at 2300 xG for approximately 30 minutes to obtain dense fibrin with platelets or a fibrin lattice. solid.

In one embodiment for carrying out the invention, another integrated system is provided for preparing a solid fibrin network as shown in Figures 11-14. The system comprises a main collection device 10, which is very similar to the main container 10 of the first embodiment. The collection device 10 may contain a density gradient cell sorting medium 26 (as described above) and an anticoagulant (not shown) as well as a reservoir 94 that can be connected to or integrated with the main collection device 10 . The above discussion relating to the exemplary embodiment, and more particularly, to the separation means 26, applies to the second embodiment of the invention. In other words, the same materials can be used for the separation medium 26, and the same materials are preferred. For example, more preferably, the separation medium 26 comprises a thixotropic gel, the yield point of which prevents it from flowing under normal ambient conditions, but allows it to

ES 2 340 705 T3 allows the higher centrifugal forces experienced during centrifugation to flow. Separation means 26 can be located at the bottom as shown in Figure 11 (ie, at the opposite end of the opening) of the main collection device. Alternatively, the separation means can form a ring around the interior of the main collection device. The main collection device 10 is essentially the same as the main container 10 described above, except that the main collection device may not contain a high density, low viscosity fluid. Preferably, the main collection device 10 has a seal 22 such as a rubber stopper or cap (as described above).

The reservoir 94 comprises a chamber 96 and a cannula 100 in fluid communication with one another. Chamber 96 contains a liquid reagent 104, more preferably a calcium clot activator. Preferably, the calcium coagulation activator is calcium chloride, calcium fluoride, calcium carbonate, calcium gluconate, calcium fumarate, calcium pyruvate, or a combination thereof. The cannula 96 must be capable of piercing the seal 22 of the main collection device 10. In a preferred embodiment, the cannula contains a blocking means 108 such as a gel with a creep limit that prevents reagents 104 in chamber 96 from flowing out of cannula 100 at ambient conditions. Other suitable locking means include, but are not limited to, force-actuated mechanical systems such as balls in springs, valves, poppet valves, pierceable membranes, and blisters (ie, hollow membranes filled with fluids or powders). The yield point of gel 108 is such that after centrifugation at a particularly high gravitational force, gel 108 moves to allow communication between chamber 96 and main collection device 10 when the two are engaged. The reservoir 94 may also have a guide housing 110 used to guide the reservoir in the collection device 10. The cannula 100 may be encompassed or covered with an elastomeric sheath 112 to maintain the sterility of the cannula 100. The sheath 112 has been discussed above with respect to the first embodiment.

In another embodiment, chamber 96 may also contain one or more of an antibiotic, an analgesic, a cancer therapeutic, a platelet growth factor, and a bone morphogenic protein. Other therapeutic agents that can be administered topically may also be included. Examples of antibiotics include, but are not limited to, ampicillin, erythromycin, and tobramycin. Pain relievers include, but are not limited to, aspirin and codeine. Cancer therapeutic agents include, but are not limited to, 5-fluoro-uracil.

In operation, a blood sample 116 from the patient is collected in the main collection device 10 by a conventional venipuncture technique as described above. The anticoagulant in the main collection device 10 dilutes the blood prior to centrifugation. Subsequently, the reservoir 94 is then attached to the main collection device 10 by piercing with the cannula 100 of the reservoir 94 through the seal 22 of the main collection device 10 as shown in Figures 13 and 14. The sleeve 112 retracts when the cannula 100 pierces seal 22. The length of cannula 100 is sufficient to pierce seal 22, but the cannula preferably does not extend much further into collection device 10, although it could.

The collection device 10 and reservoir 94 are then centrifuged. The centrifugal force exerted on the tube is described by the equation F = wmr²; where F = force, m = mass of the system, r = radial distance from the center of the rotor, and ω = the angular rotation speed. Since the reservoir is at a smaller r than the gel in the main tube, the gel in the reservoir cannula cannot move since insufficient shear stress is generated. The main tube 10 rotates at the low gravitational force until the cells separate and the gel 26 moves to the cell / plasma interface as shown in Figure 13. In other words, similar to the first embodiment, Separation medium 26 separates red blood cells 30 from platelet rich plasma 34 after initial centrifugation at about 1000 xG for about 10 minutes. Centrifugation at a centrifugal force of approximately 900-1500 xG for approximately 5 to 15 minutes is also acceptable for initial centrifugation.

Subsequently, the centrifuge speed is increased and the reservoir experiences a sufficiently high gravitational force so that the locking means 108 in the cannula 100 is emptied into the main collection device 10 and the liquid reagent 108 (for example, the activator of the calcium coagulation) is emptied from the reservoir as shown in Figure 14. The contents can then be centrifuged at about 2300-6000 xG for about 15-40 minutes. As the calcium clotting activator comes into contact with the plasma in the main collection device, immediate and simultaneous clotting and centrifugation occurs because the sample is still being centrifuged. This causes the formation of a solid fibrin network suitable for tissue regeneration. The cell separation operation in the main tube and the subsequent addition of the liquid coagulating agent at the appropriate stoichiometric ratio is carried out in a tube without transfer. By programming the centrifuge with regard to speed and duration, the invention provides a simple and foolproof process.

In an alternative embodiment, the single collection device 10 has an interior compartment 119 and a reservoir 94 as shown in Figures 15-16. The reservoir 94 is integrated with or connected to the main collection device 10 and in fluid communication with the compartment. A tube, conduit, or opening 120 provides fluid communication between compartment 119 and reservoir 94, and is sealed with locking means 108. Again, the locking means 108 has a yield point that activates and moves when exposed to a particularly high gravitational force to allow communication between the reservoir 94 and the main collection device 10 as described above. The yield point of the gel or medium is such that it does not move during the initial centrifugation to separate red blood cells from the plasma. In the third embodiment, each end of the device has an opening and each end is hermetically sealed by a removable seal.

ES 2 340 705 T3 or non-removable 22, 122 such as a rubber stopper, cap, foam, elastomer or other compound. Reservoir 94 with cap 122 is located at the opposite end of seal 22 and the opening of the collection device.

In another embodiment, reservoir 94 may also contain one or more of an antibiotic, an analgesic, a cancer therapeutic, a platelet growth factor, and a bone morphogenic protein. Other therapeutic agents that can be administered topically may also be included. Examples of antibiotics include, but are not limited to, ampicillin, erythromycin, and tobramycin. Pain relievers include, but are not limited to, aspirin and codeine. Cancer therapeutic agents include, but are not limited to, 5-fluoro-uracil.

The alternative embodiment is used in the same way as described above with respect to the first mentioned embodiment, i.e. the centrifuge is controlled at two different centrifugal forces: 1) the first being a sufficient force to separate red blood cell plasma; and 2) the second being a sufficient force to move the locking means 108 in the tube, conduit or opening 120 between the reservoir and the interior of the device and in the main body. As a result, the calcium clotting activator is allowed to enter the interior of the device. This in turn enables simultaneous centrifugation and coagulation of the plasma to form the solid fibrin network as centrifugation continues at the second highest gravitational force. The seal 122 can be removed to obtain the solid fibrin network or the autologous glue. In a preferred embodiment, the seal 122 is threaded and can be unscrewed from the device 10 as shown in Figure 16.