Abstract:
A sterile method for preparing stable thrombin component from a single donor&#39;s plasma in which the thrombin component and the clotting and adhesive proteins component are harvested simultaneously from the same donor plasma in less than one hour. The combined components provide an improved biological hemostatic agent and tissue sealant by virtue of its freedom from the risk of contaminating viruses or bacteria from allogenic human or bovine blood sources. The thrombin provides polymerization of the clotting and adhesive proteins in less than five seconds, and is sufficiently stable to provide that fast clotting over a six hour period. Further, the clotting times can be predictably lengthened by diluting the thrombin with saline.

Description:
FIELD OF THE INVENTION 
     The following invention relates generally to the preparation of a high specific activity thrombin enzyme from a given unit of plasma, which is sufficiently stable that it provides rapid clotting of a fibrinogen-rich solution of clotting and adhesive proteins for more than six hours when held at room temperature or lower. 
     BACKGROUND OF THE INVENTION 
     Formulation of a fibrin sealant mimics the last step of the coagulation cascade wherein the enzyme thrombin cleaves fibrinogen which is then cross-linked into a semi-rigid or flexible fibrin clot. This fibrin clot adheres to wound sites, forming a barrier to fluid leaks and generates adhesion between tissues, while providing hemostatic and healing properties to the treated site. 
     Presently marketed, applicant&#39;s CryoSeal™ system is a device which harvests cryoprecipitated, concentrated clotting and adhesive proteins, including fibrinogen and Factor XIII from a donor&#39;s plasma in approximately one hour. The one hour cryoprecipitation harvesting, provided by the CryoSeal™ system, compared to the 1 to 2 day cryoprecipitation process routinely practiced in Blood Banks, means that CryoSeal™ harvesting of clotting and adhesive proteins can occur right in the perioperative theater with the patient close by, thereby avoiding the need to initiate the process days in advance. 
     These CryoSeal™ harvested clotting and adhesive proteins, when combined with bovine or human thrombin, forms a biological glue useful for surgical hemostasis and tissue adhesion. Commercially available thrombin, however, is generally sourced from bovine or human plasma pools, so the patient would still be at risk of negative immune reactions or contamination by infectious blood born viruses and, possibly Crutzfeld-Jacobs Disease (CJD) or new variants of CJD (NVCJD). An advantage of the CryoSeal™ cryoprecipitation invention is that the harvested clotting and adhesive proteins sourced from the patient&#39;s own blood eliminates the risk of contamination by infectious blood-borne disease when these clotting and adhesive proteins are topically applied to the patient&#39;s surgical wound sites. 
     It has long been understood, however, that the safest condition for a surgical patient would result from a two component biological sealant preparation in which the thrombin component would be harvested from the same donor in which the clotting and adhesive protein component was harvested—forming a fully autologous biological sealant or glue. 
     The concept of utilizing thrombin and/or fibrinogen sourced from the patient in a medical procedure performed on that patient is not novel and was first described by Andrianova in 1974. Some twenty years later, Cederholm-Williams PCT Patent (WO94/00566—Jan. 6, 1994) describes an improved fibrin glue in which the thrombin component, which required thirteen steps, including centrifugation, and separation of intermediate precipitates and adjusting the ionic strength of the blood and pH of the plasma to prepare, would be combined with a fibrinogen component also sourced from the plasma of the same donor. However, these many preparation steps are so time consuming they become impractical for use in the perioperative theater where processing times should be less than one hour. 
     Three years later, in 1997, Hirsh, et al. (U.S. Pat. No. 5,643,192) follows Cederholm-Williams by teaching another method of preparing fibrin glue in which both the fibrinogen and thrombin components of a fibrin glue are sourced from the same donor&#39;s plasma. The Hirsh patent describes a method of preparing thrombin in which most of the fibrinogen in the plasma is first precipitated and removed to prepare a supernatant and then clotting the residual fibrinogen in the supernatant which is different and simpler than the method taught by Cederholm-Williams, but does not result in a commercially useful thrombin in that (see FIG. 1 of Hirsh, et al.) the thrombin provides clotting speeds of five seconds or less for only 4 minutes, and less than 10 seconds for only 47 minutes. 
     These clotting speeds are unsuitable to the needs of surgeons who could not plan their entire surgeries around the limitations of the Hirsh, et al. fibrin glue. 
     Surgeons predominately require a fast acting clotting time (&lt;5 seconds) for hemostasis and tissue sealing or adhesion. Slow clotting biological glues (&gt;5 seconds) will often be transported away from the wound site by oozing and bleeding before they can perform their function. A surgeon utilizing the Hirsh fibrin glue would be required to arrange his surgery so that the hemostasis and tissue sealing intended for treatment with the Hirsh fibrin glue would occur within the 4 minute window where the clotting time was less than 5 seconds, making the Hirsh invention totally impractical for most surgeries which predominantly require rapid hemostasis and tissue adhesion throughout the surgery, the time span of which could extend to six hours. 
     The following prior art reflects the state of the art of which applicant is aware and is included herewith to discharge applicant&#39;s acknowledged duty to disclose relevant prior art. It is stipulated, however, that none of these references teach singly nor render obvious when considered in any conceivable combination the nexus of the instant invention as disclosed in greater detail hereinafter and as particularly claimed. 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
             
             
               
                 U.S. PATENT DOCUMENTS 
               
             
          
           
               
                 INVENTOR 
                 U.S. PAT. NO. 
                 ISSUE DATE 
               
               
                   
               
             
          
           
               
                 Pumphrey 
                   713,017 
                 November 4, 
                 1902 
               
               
                 Mobley 
                 1,614,532 
                 January 18, 
                 1927 
               
               
                 Ferry, et al. 
                 2,533,004 
                 December 5, 
                 1950 
               
               
                 Wahlin 
                 2,747,936 
                 May 29, 
                 1956 
               
               
                 Clark 
                 3,179,107 
                 April 20, 
                 1965 
               
               
                 Cobey 
                 3,223,083 
                 December 14, 
                 1965 
               
               
                 Kennedy, et al. 
                 3,236,457 
                 February 22, 
                 1966 
               
               
                 Meurer, et al. 
                 3,269,389 
                 August 30, 
                 1966 
               
               
                 Venus, Jr. 
                 3,416,737 
                 December 17, 
                 1968 
               
               
                 Horn 
                 3,467,096 
                 September 16, 
                 1969 
               
               
                 Creighton, et al. 
                 3,828,980 
                 August 13, 
                 1974 
               
               
                 Green 
                 3,942,725 
                 March 9, 
                 1976 
               
               
                 Polnauer, deceased, et al. 
                 3,945,574 
                 March 23, 
                 1976 
               
               
                 Speer 
                 4,040,420 
                 August 9, 
                 1977 
               
               
                 Reinhardt, et al. 
                 4,067,333 
                 January 10, 
                 1978 
               
               
                 Kozam, et al. 
                 4,109,653 
                 August 29, 
                 1978 
               
               
                 Sugitachi, et al. 
                 4,265,233 
                 May 5, 
                 1981 
               
               
                 Schwarz, et al. 
                 4,298,598 
                 November 3, 
                 1981 
               
               
                 Redl, et al. 
                 4,359,049 
                 November 16, 
                 1982 
               
               
                 Schwarz, et al. 
                 4,362,567 
                 December 7, 
                 1982 
               
               
                 Altshuler 
                 4,363,319 
                 December 14, 
                 1982 
               
               
                 Schneider 
                 4,374,830 
                 February 22, 
                 1983 
               
               
                 Schwarz, et al. 
                 4,377,572 
                 March 22, 
                 1983 
               
               
                 Schwarz, et al. 
                 4,414,976 
                 November 15, 
                 1983 
               
               
                 Stroetmann 
                 4,427,650 
                 January 24, 
                 1984 
               
               
                 Stroetmann 
                 4,427,651 
                 January 24, 
                 1984 
               
               
                 Stroetmann 
                 4,442,655 
                 April 17, 
                 1984 
               
               
                 Zimmerman, et al. 
                 4,453,939 
                 June 12, 
                 1984 
               
               
                 Rose, et al. 
                 4,627,879 
                 December 9, 
                 1986 
               
               
                 Redl, et al. 
                 4,631,055 
                 December 23, 
                 1986 
               
               
                 Sakamoto, et al. 
                 4,655,211 
                 April 7, 
                 1987 
               
               
                 Silbering, et al. 
                 4,696,812 
                 September 29, 
                 1987 
               
               
                 Alterbaum 
                 4,714,457 
                 December 22, 
                 1987 
               
               
                 Koizumi, et al. 
                 4,734,261 
                 March 29, 
                 1988 
               
               
                 Eibl, et al. 
                 4,735,616 
                 April 5, 
                 1988 
               
               
                 Saferstein, et al. 
                 4,752,466 
                 June 21, 
                 1988 
               
               
                 Wolf, et al. 
                 4,767,416 
                 August 30, 
                 1988 
               
               
                 Skorka, et al. 
                 4,826,048 
                 May 2, 
                 1989 
               
               
                 Davis 
                 4,842,581 
                 June 27, 
                 1989 
               
               
                 Miller, et al. 
                 4,874,368 
                 October 17, 
                 1989 
               
               
                 Avoy 
                 4,902,281 
                 February 20, 
                 1990 
               
               
                 Seelich 
                 4,909,251 
                 March 20, 
                 1990 
               
               
                 Tanaka, et al. 
                 4,923,815 
                 May 8, 
                 1990 
               
               
                 Silbering, et al. 
                 4,965,203 
                 October 23, 
                 1990 
               
               
                 Capozzi, et al. 
                 4,978,336 
                 December 18, 
                 1990 
               
               
                 Wolf, et al. 
                 4,979,942 
                 December 25, 
                 1990 
               
               
                 L&#39;Hermite, et al. 
                 4,987,336 
                 January 22, 
                 1991 
               
               
                 La Duca 
                 5,089,415 
                 February 18, 
                 1992 
               
               
                 Kotitschke, et al. 
                 5,099,003 
                 March 24, 
                 1992 
               
               
                 Wolf, et al. 
                 5,104,375 
                 April 14, 
                 1992 
               
               
                 Capozzi, et al. 
                 5,116,315 
                 May 26, 
                 1992 
               
               
                 Nishimaki, et al. 
                 5,130,244 
                 July 14, 
                 1992 
               
               
                 Kraus, et al. 
                 5,143,838 
                 September 1, 
                 1992 
               
               
                 Crowley, et al. 
                 5,151,355 
                 September 29, 
                 1992 
               
               
                 Knighton 
                 5,165,938 
                 November 24, 
                 1992 
               
               
                 Galanakis 
                 5,185,001 
                 February 9, 
                 1993 
               
               
                 Morse, et al. 
                 5,219,328 
                 June 15, 
                 1993 
               
               
                 Fischer 
                 5,290,259 
                 March 1, 
                 1994 
               
               
                 Sierra, et al. 
                 5,290,552 
                 March 1, 
                 1994 
               
               
                 Michalski, et al. 
                 5,304,372 
                 April 19, 
                 1994 
               
               
                 Fischer 
                 5,328,462 
                 July 12, 
                 1994 
               
               
                 Lonneman, et al. 
                 5,368,563 
                 November 29, 
                 1994 
               
               
                 Linnau 
                 5,393,666 
                 February 28, 
                 1995 
               
               
                 Epstein 
                 5,405,607 
                 April 11, 
                 1995 
               
               
                 Marx 
                 5,411,885 
                 May 2, 
                 1995 
               
               
                 Kikuchi, et al. 
                 5,443,959 
                 August 22, 
                 1995 
               
               
                 Miller, et al. 
                 5,474,540 
                 December 12, 
                 1995 
               
               
                 Broly, et al. 
                 5,474,770 
                 December 12, 
                 1995 
               
               
                 Weis-Fogh, et al. 
                 5,480,378 
                 January 2, 
                 1996 
               
               
                 Proba, et al. 
                 5,506,127 
                 April 9, 
                 1996 
               
               
                 Cochrum 
                 5,510,102 
                 April 23, 
                 1996 
               
               
                 Antanavich, et al. 
                 5,585,007 
                 December 17, 
                 1996 
               
               
                 Pines, et al. 
                 5,605,887 
                 February 25, 
                 1997 
               
               
                 Cochrum 
                 5,614,204 
                 March 25, 
                 1997 
               
               
                 Marx 
                 5,631,019 
                 May 20, 
                 1997 
               
               
                 Hirsh, et al. 
                 5,643,192 
                 July 1, 
                 1997 
               
               
                 Epstein 
                 5,648,265 
                 July 15, 
                 1997 
               
               
                 Edwardson, et al. 
                 5,750,657 
                 May 12, 
                 1998 
               
               
                 Cederholm-Williams 
                 5,795,571 
                 August 18, 
                 1998 
               
               
                 Cederholm-Williams 
                 5,795,780 
                 August 18, 
                 1998 
               
               
                 Edwardson, et al. 
                 5,804,428 
                 September 8, 
                 1998 
               
               
                   
               
             
          
           
               
                 FOREIGN PATENT DOCUMENTS 
               
             
          
           
               
                 APPLICANT 
                 COUNTRY 
                 PATENT NO. 
                 ISSUE DATE 
               
               
                   
               
             
          
           
               
                 Zdaril 
                 DE 
                 DE 25,913 
                 February 12, 
                 1884 
               
               
                 Szent-Györgyi, 
                 CH 
                 259,254 
                 June 1, 
                 1949 
               
               
                 et al. 
               
               
                 The Trustees of 
                 WIPO 
                 WO 86/01814 
                 March 27, 
                 1986 
               
               
                 Columbia Univer- 
               
               
                 sity in the City 
               
               
                 of New York 
               
               
                 Weis-Fogh 
                 WIPO 
                 WO 88/02259 
                 April 7, 
                 1988 
               
               
                 Board of Regents, 
                 WIPO 
                 WO 88/03151 
                 May 5, 
                 1988 
               
               
                 The University of 
                 SU 
                 1,527,261 A1 
                 December 7, 
                 1989 
               
               
                 Texas System 
               
               
                 Cryolife, Inc. 
                 WIPO 
                 WO 91/09641 
                 July 11, 
                 1991 
               
               
                 Baxter Inter- 
                 EP 
                 0 443 724 A1 
                 August 28, 
                 1991 
               
               
                 national, Inc. 
               
               
                 Warner- 
                 EP 
                 0 505 604 A1 
                 September 30, 
                 1992 
               
               
                 Lambert Co. 
               
               
                 Octapharma AG 
                 EP 
                 0 534 178 A2 
                 March 31, 
                 1993 
               
               
                 Cryolife, Inc. 
                 WIPO 
                 WO 93/19805 
                 October 14, 
                 1993 
               
               
                 Cederholm- 
                 WIPO 
                 WO 94/00566 
                 January 6, 
                 1994 
               
               
                 Williams, et al. 
               
               
                 E. R. Squibb &amp; 
                 EP 
                 0 592 242 A1 
                 April 13, 
                 1994 
               
               
                 Sons 
               
               
                 Plasmaseal 
                 WIPO 
                 WO 96/17871 
                 June 13, 
                 1996 
               
               
                 Corporation 
               
               
                   
               
             
          
         
       
     
     OTHER PRIOR ART (INCLUDING AUTHOR, TITLE, PERTINENT PAGES, DATE, ETC.) 
     Fenton, J. W., et al., “Human Thrombins”, Chemistry &amp; Biology of Thrombin, pp. 43-70. 
     Rosenberg, R. D., et al., “Bovine Thrombin: Constant Specific Activity Products From Single Animals”, Fed. Proc., p. 321, Abstract No. 361. 
     Quick, A. J., et al., “Production Of Thrombin From Precipitate Obtained By Acidification Of Diluted Plasma”, pp. 114-118. 
     Eagle, H., “Studies On Blood Coagulation”, pp. 531-545, 1934. 
     Mann, K. G., et al., “The Molecular Weights Of Bovine Thrombin And Its Primary Autolysis Products”, pp. 6555-6557, 1969. 
     Mann, K. G., et al., “Multiple Active Forms Of Thrombin”, pp. 5994-6001, 1971. 
     Martin, M., et al., “Thrombolysis In Patients With Chronic Arterial Occlusions”, Thrombolytic Therapy, Vol. 47, pp. 235-241, 1971. 
     Fenton, J. W., et al., “Large-Scale Preparation And Preliminary Characterizations Of Human Thrombin”, Biochimica et Biophysica Acta. Vol. 229, pp. 26-32, 1971. 
     Andrianova, et al., “An Accessible Method Of Simultaneous Production Of Fibrinogen And Thrombin From Blood”, pp. 648-650, 1975. (Plus English translation). 
     Georgi, M., et al., “Occlusion Of The Renal Artery By Intra-Arterial Injection Of Thrombin In A Case Of Inoperable Renal Tumor”, Deutsche Medizinische Wochenschrift, Vol. 100(47), pp. 2428-2429, 1975. (Plus English translation). 
     Lundblad, R. L., et al., “Preparation And Partial Characterization Of Two Forms Of Bovine Thrombin”, Biochemical and Biophysical Research Communications, Vol. 66(2), pp. 482-489, 1975. 
     Sakuragawa, N., et al., “Purification And Some Characterization Of Human Thrombin”, Acta Medica et Biologica, Vol. 23(1), pp. 65-73, 1975. 
     Fenton, J. W., et al., “Human Thrombins: Production, Evaluation, And Properties Of α-Thrombin”, The Journal of Biological Chemistry, Vol. 252(11), pp. 3587-3598, 1977. 
     Nordenman, B., et al., “Purification Of Thrombin By Affinity Chromatography On Immobilized Heparin”, Thrombosis Research, Vol. 11, pp. 799-808, 1977. 
     Nowotny, R., et al., “Mechanical Properties Of Fibrinogen-Adhesive Material”, Biomaterials 1980, Vol. 3, pp. 677-682, 1982. 
     Kotelba-Witkowska, B., et al., “Cryopreservation Of Platelet Concentrates Using Glycerol-Glucose”, Transfusion, Vol. 22(2), pp. 121-124, 1982. 
     Redl, H., et al., “Fibrin Sealant-Antibiotic Mixture—Stability And Elution Behavior”, Fibrinkleber Orthop. Traumatol. Orthop. Symp., Vol. 4, pp. 178-181, 1982. (Plus English translation). 
     Redl, H., et al., “In Vitro Properties Of Mixtures Of Fibrin Seal And Antibiotics”, Biomaterials, Vol. 4(1), pp. 29-32, 1983. 
     Gestring, G., et al., “Autologous Fibrinogen For Tissue-Adhesion, Hemostasis And Embolization”, Vascular Surgery, Vol. 17, pp. 294-304, 1983. 
     Wolf, G., “The Concentrated Autologous Tissue Glue”, Archives of Oto-Rhino-Laryngology, Vol. 237, pp. 279-283, 1983. 
     Tsvetkov, T. S., et al., “A Method For Preparation Of Dry Thrombin For Topical Application”, Cryobiology, Vol. 21(6), pp. 661-663, 1984. 
     Yu, X. J., et al., “Affinity Chromatography Of Thrombin On Modified Polystyrene Resins”, Journal of Chromatography, Vol. 376, pp. 429-435, 1986. 
     Fischer, A. M., et al., “Thrombin Purification By One-Step Preparative Affinity Chromatography On Modified Polystyrenes”, Journal of Chromatography, Vol. 363(1), pp. 95-100, 1986. 
     Harpel, P. C., “Blood Proteolytic Enzyme Inhibitors: Their Role In Modulating Blood Coagulation And Fibrinolytic Enzyme Pathways”, pp. 219-234, 1987. 
     Fenton, J. W., “Regulation Of Thrombin Generation And Functions”, Seminars in Thrombosis and Hemostasis, Vol. 14(3), pp. 234-240, 1988. 
     Awano, K., et al., “Role Of Seratonin, Histamine, And Thromboxane A 2  In Platelet-Induced Contractions Of Coronary Arteries And Aortae From Rabbits”, Journal Of Cardiovascular Pharmacology, Vol. 13(5), pp. 781-792, 1989. 
     Mulvihill, J., et al., “Thrombin Stimulated Platelet Accumulation On Protein Coated Glass Capillaries: Role Of Adhesive Platelet α-Granule Proteins”, Thrombosis and Haemostasis, Vol. 62(3), pp. 989-995, 1989. 
     Suzuki, S., et al., “A Study On The Properties Of Commercial Thrombin Preparations”, Thrombosis Research, Vol. 53(3), pp. 271-277, 1989. 
     Ronfard, V., et al., “Use of Human Keratinocytes Cultured On Fibrin Glue In The Treatment Of Burn Wounds”, Burns, Vol. 17(3), pp. 181-184, 1991. 
     Brennan, M., “Fibrin Glue”, Blood Reviews, Vol. 5, pp. 240-244, 1991. 
     DePalma, L., et.al., “The Preparation Of Fibrinogen Concentrate For Use As Fibrin Glue By Four Different Methods”, Transfusion, Vol. 33(9), pp. 717-720, 1993. 
     McCarthy, P., “Fibrin Glue In Cardiothoracic Surgery”, Transfusion Medicine Reviews, Vol. 7(3), pp. 173-179, 1993. 
     Cederholm-Williams, S., “Benefits Of Adjuvant Fibrin Glue In Skin Grafting”, The Medical Journal of Australia, Vol. 161(9), p. 575, 1994. 
     Cederholm-Williams, S., “Autologous Fibrin Sealants Are Not Yet Available”, The Lancet, Vol. 344, p. 336, 1994. 
     Wiegand, D. A., et al., “Assessment Of Cryoprecipitate-Thrombin Solution for Dural Repair”, Head &amp; Neck, pp. 569-573, 1994. 
     The other prior art listed above, not all of which are specifically discussed catalog the prior art of which the applicant is aware. These undiscussed references diverge even more starkly from the instant invention specifically distinguished below. 
     SUMMARY OF THE INVENTION 
     The instant invention addresses the long felt need for a simple, practical, fast method of preparing stable human thrombin from a donor&#39;s blood, which will provide fast clots (&lt;5 seconds) throughout a lengthy surgery (e.g. six hours) to combine with the clotting and adhesive proteins harvested and concentrated from the same unit of blood to form a biological sealant with no patient exposure to microbial or possible CJD or NVCJD contaminations. Previous works in the field (Hirsch, et al.) exemplified a thrombin with minimal stability in that the thrombin achieved rapid clotting of fibrinogen (i.e., less than 5 seconds) during only a very narrow four to five minute time period, or required so many steps and elapsed time it would not be suitable for perioperative preparation, both totally impractical for the broad range of surgeries. 
     The present invention provides a stable thrombin enzyme which can cause precise, repeatable fast or slow polymerization of clotting and adhesive proteins over a duration of up to six hours—throughout even a long surgery. Further, the use of clotting and adhesive proteins and thrombin all sourced from a single donor will eliminate various disease risks posed from the use of commercial fibrin glues where the fibrinogen is sourced from plasma pooled from thousands of donors and the thrombin is either sourced from a similar pool of human plasma or of bovine origin. The speed and simplicity of the production of stable thrombin by use of this invention allows it to be prepared just prior to or during operative procedures and it will provide fast clotting throughout even the longest surgeries. The thrombin produced by this invention can be diluted in saline, water and a dilute CaCl 2  solution (e.g. 125 mM CaCl 2 ) to provide precise, slower clotting times thereby allowing any preferred time from less than five seconds to longer than 2 minutes. 
     The procedure of the invention is preferably comprised of three steps, the first two of which should preferably occur at the same time: 
     1. Preparing a fraction enriched in prothrombin by use of Ethanol to substantially enhance the concentration of prothrombin and at the same time remove or denature naturally occurring ingredients within plasma, such as Fibrinogen and Antithrombin III which can bind to, block, interfere with or inhibit prothrombin or its subsequent activation to long-term functional thrombin. 
     2. Adding calcium ions to the enriched prothrombin solution and briefly agitating the solution to convert the pro-thrombin to stable, long term thrombin. 
     3. Expressing the thrombin solution through a filter to remove particulate matter which would prevent spraying the thrombin through a small orifice or expressing the thrombin through a thin tube onto a wound site. 
     OBJECTS OF THE INVENTION 
     Accordingly, it is a primary object of the present invention to provide a new and novel apparatus and method to derive fast acting, stable autologous thrombin from the donor&#39;s plasma. 
     It is a further object of the present invention to provide thrombin as characterized above which has a shelf life longer than most associated surgical procedures. 
     It is a further object of the present invention to provide thrombin as characterized above in which the clotting time can be predictably lengthened at will through dilution with saline. 
     It is a further object of the present invention to provide thrombin as characterized above which has simple preparatory procedures. 
     It is a further object of the present invention to provide a method for producing thrombin as characterized above which has a process time in as little as thirty minutes, up to seventy-five minutes. 
     It is a further object of the present invention to provide thrombin which can be sprayed through small orifices or expressed through thin tubes. 
     Viewed from a first vantage point it is the object of the present invention to provide a novel and practical method for producing stable human thrombin from a prothrombin fraction which has been substantially enriched by ethanol fractionation to increase the prothrombin concentration and at the same time remove contaminating proteins. The addition of calcium chloride to the enriched prothrombin converts prothrombin to thrombin. From the same sole donor plasma, clotting and adhesive proteins are simultaneously obtained by other means to comprise the second component necessary for the autologous biological sealant. 
     Viewed from a second vantage point, it is an object of the present invention to provide a method for generating autologous thrombin from a patient, the steps including: obtaining a blood product from the patient; sequestering plasma from the product; enriching the prothrombin in a plasma fraction; converting the prothrombin to thrombin, and filtering particulate from the thrombin. 
     Viewed from a third vantage point, it is an object of the present invention to provide a method for producing autologous thrombin which is stable for more than fifteen minutes, the steps including: sequestering pro-thrombin from plasma and converting the pro-thrombin to thrombin. 
     Viewed from a fourth vantage point, it is an object of the present invention to provide an autologous thrombin which provides fast clotting in less than five seconds for more than fifteen minutes. 
     Viewed from a fifth vantage point, it is an object of the present invention to provide a composition for extracting thrombin from plasma consisting essentially of: Plasma; Ethanol (ETOH); CaCl 2 . 
     Viewed from a sixth vantage point, it is an object of the present invention to provide a method for preparing thrombin comprising: obtaining plasma; adding ETOH and CaCl 2  to the plasma, forming a composition: agitating the composition; incubating the composition in a static or rocking mode; filtering the composition of particulate, thereby passing the thrombin through the filter. 
     Viewed from a seventh vantage point, it is an object of the present invention to provide a device for preparing thrombin from plasma, comprising: a reaction chamber having a solution of CaCl 2  and ETOH therein; means for admitting plasma into the reaction chamber; thrombin receiving syringe coupled to the reaction chamber to receive the thrombin; and a filter located between the reaction chamber and the thrombin receiving syringe. 
     Viewed from an eighth vantage point, it is an object of the present invention to provide an autologous biological glue processing device, comprising, in combination: a thrombin processing means, a clotting and adhesive proteins processing means operatively coupled to the thrombin processing means, means for receiving plasma via the operative coupling for subsequent conversion of the plasma to, respectively thrombin and clotting and adhesive proteins. 
     The present invention provides a method and apparatus that produces thrombin which is sufficiently stable that it can provide less-than-5-second clots for up to six hours, substantially more stable than demonstrated in all prior art. Further, the clot time can be modified at will through dilution with saline. 
     The present invention further provides an efficient method of preparation. Improved cryoprecipitation of clotting and adhesive proteins through the CryoSeal™ invention requires less than one hour. In this same time frame, the autologous human thrombin component can be manufactured with minimal materials and methods from the same source plasma. Both of the biological components of the biological glue are easily combined in a surgical setting, administered to the very same donor patient, and the resultant clotting provides hemostasis or tissue adhesion at the wound site. 
     The present invention additionally provides a method for sterile production of both components of the biological glue. The improved sterile manufacturing described herein provides a final product that is essentially free of contamination by non autologous microbes. 
     These and other objects will be made manifest when considering the following detailed specification when taken in conjunction with the appended drawing figures. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a perspective view of an apparatus for sequestering prothrombin from plasma, processing the prothrombin into thrombin and taking the plasma not relegated towards the prothrombin and extracting clotting and adhesive proteins therefrom. 
     FIG. 2 is a plan view of the thrombin processing set removed from the processing set that extracts clotting and adhesive proteins. 
     FIG. 3 is a perspective view of the interior of the thrombin processing case with the thrombin syringe shown in FIG. 2 removed therefrom. 
     FIG. 4 is a perspective view of the thrombin case upper half. 
     FIG. 5 is a perspective view of the thrombin case lower half. 
     FIG. 6 is an exploded parts view of the reaction chamber  26  shown in FIG. 3 along with the valving structure at opposed ends thereof. 
     FIG. 7 is a sectional view of the reaction chamber and valving structure depicted in FIG.  6 . 
     FIG. 8 is a detail of construction of one component of that which is shown in FIG.  7 . 
     FIG. 9 is an exploded parts view of a filter shown in FIG.  3 . 
     FIG. 10 is a perspective view of that which is shown in FIG.  9 . 
     FIG. 11 graphs clot time versus lifespan of thrombin fractionated at different ETOH concentrations. 
     FIG. 12 graphs clot time versus lifespan of thrombin fractionated at different ETOH concentrations at different CaCl 2  concentrations. 
     FIG. 13 graphs clot time versus lifespan of thrombin showing reagent volume sensitivity when the thrombin is stored on ice. 
     FIG. 14 graphs clot time versus lifespan of thrombin showing reagent volume sensitivity when the thrombin is stored at room temperature. 
     FIG. 15 graphs clot time versus lifespan of thrombin showing plasma volume sensitivity when the thrombin is stored on ice. 
     FIG. 16 graphs clot time versus lifespan of thrombin showing plasma volume sensitivity when the thrombin is stored at room temperature. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Referring to the drawings, wherein like elements denote like parts throughout, reference numeral  10  is directed to the processing set according to the present invention and shown in FIG.  1 . 
     In its essence, the processing set  10  includes a fluid receiving system  20  which communicates with both a thrombin processing unit  40  and a clotting and adhesive proteins processing unit  60 . 
     More particularly, the fluid receiving system  20  includes an inlet  2  communicating with tubing  4  through which plasma will enter the processing units  40 ,  60 . The conduit  4  has plural stop valves  6  which can occlude the tubing  4  preventing fluids through passage. The tubing  4  communicates through a T fitting  8  to divide plasma into two branches, a first branch  12  which leads to the thrombin processing unit  40  and a second branch  14  leading to the clotting and adhesive proteins processing unit  60 . The first valve branch  12  also includes a stop valve  6 . 
     Since it is preferred that the blood product admitted to the inlet  2  be plasma, the whole blood is first processed either by filtering, centrifugation, or another means of settling to remove the heavier red blood cells from the blood products, leaving plasma therebeyond for use in the FIG. 1 device. Although this system can be dimensioned for any size batch, the plasma required for the thrombin processing unit will typically be 9-10 ml. so that the final volume of concentrated thrombin matches a typical yield of cryoprecipitated clotting and adhesive proteins from the clotting and adhesive proteins processing unit  60 . A sealed bag  16  overlies the thrombin dispensing syringe  42  (and a lead in of conduit  64 ) to provide sterility until the thrombin dispensing syringe  42  is introduced into a sterile surgical field (e.g., operatory). 
     Prior to that, the thrombin processing unit  40  operates as shown and described with reference to FIGS. 2 through 10. As mentioned, fluid enters the first branch  12  and (FIG. 1) passes beyond a coupling  18  and into an interior of a casing  22 . Coupling  18  is preferably frictionally and/or adhesively attached to the first branch  12  yet the thrombin processing unit  40  can still be removed (e.g. FIG. 2) from the processing set  10  (e.g., by merely detaching or severing branch  12  followed perhaps with heat sealing) after receiving the plasma as shown in FIG.  2 . If adhesive is used, it is a sterile grade for use in an operatory. 
     Referring to FIG. 3, a valve  24  initially directs the plasma to a reaction chamber  26  having an interior tube  28  (FIG. 6) preferably formed from glass and capable of receiving a volume, for example 15 ml. Glass tube  28  is preferably shorter than and circumscribed by an overlying barrel  32  preferably formed from PVC. A window  31  in the PVC barrel  32  can be used to gauge and/or verify the contents within the glass tube  28 . Gauging may also include gradations  29 , indicating a volume on the glass tube. The glass tube  28  of the reaction chamber  26  receives the plasma from the first branch  12  and into its interior for mixing with reagents preloaded in the glass tube  28  and described hereinafter. As shown in FIG. 7, the interior of the glass tube is preferably prefilled only partially with beads  25  preferably formed from borosilicate to enhance the reaction and agitation. 
     The reaction chamber  26  is formed with first and second end caps  34  detailed in FIGS. 6 through 8. Each end cap includes a central outwardly conically tapering spout  36  which communicates with the valve  24  at one end and a further valve  44  at an opposite end. Each spout  36  is isolated from the beads  25  by a screen  23  nested within necked-down portion  48 . Valve  24  has three branches as does valve  44 , but valve  44  has one branch capped off with a cap  45  thereby defining a two branch valve. One branch of each valve  24 ,  44  communicates with a respective one spout  36  projecting out from each cap  34 . Fluid communication exists between one branch of each valve and its spout into the interior of the glass tube  28  and through flow is controlled by the valves  24 ,  44 . As shown in FIG. 8, the cap  34  includes an annular necked-down portion  48  which frictionally and/or adhesively resides within an interior hollow of the PVC barrel  32 . In this way, the necked-down portion  48  rests upon ends of the glass tube  28  in sealing engagement therewith, isolating the interior of the reaction chamber from the PVC barrel  32 . 
     Preferably, ethanol and calcium chloride are the reagents which have been preloaded into the reaction chamber  26 . Initially, both valves  24  and  44  are oriented so that reagents will not pass therebeyond to seal the chamber. After the plasma has been pumped into processing unit  60 , valve  44  is turned to allow access to the draw plunger  56  and valve  24  is oriented to allow access between the passageway  21  and the reaction chamber  26 . Slide clip  6  is opened with the thrombin processing unit  40  held vertically with respect to the plan shown in FIG. 1, syringe  56  plunger  58  is moved along the direction of the arrow A to evacuate air from chamber  26 . More specifically, the path  43  between valve  44  and syringe  56  includes a filter  62  located in the flow path. The filter  62  provides an aesceptic microbial barrier so that, upon subsequent delivery of the thrombin to the dispensing syringe  42  (FIG.  1 ), there is no contamination from around the seal  57  of plunger  58  delivered to syringe  42 . Plasma will subsequently enter chamber  26  from conduit  4  to replace air. Valve  24  is oriented to address filter  66 . The reagents and plasma are briefly agitated assisted by beads  25  (and allowed to incubate for about 60 minutes). After incubation, thrombin processing unit  40  is agitated to loosen and break up gel formation. The plunger of syringe  56  is pushed in the direction opposite arrow A to move thrombin from chamber  26  through filter  66  into syringe  42 . Delivery of thrombin to syringe  42  can be enhanced by retracting plunger  43  of syringe  42 , defining a push pull system. Filter  66  removes particulate matter from the thrombin, including gel. 
     FIGS. 9 and 10 reveal the filter  66  includes an outer cylindrical wall  65  with end caps  34  each having a cylindrical spout  37  circumscribed by an annular recess  39 . The centrally disposed cylindrical filter element  67  is preferably formed from polyurethane foam. Filter  67  filters by weight, size and protein binding. 
     Allowing the thrombin contained in the reaction chamber  26  to reside therein after agitation for 30 to 75 (until a gel formation occurs in the reaction chamber) enhances the effectiveness of the filter  66  in removing particulate matter for subsequent utilization. The time span for conversion and activation allows enough particulate matter to be removed by the filter to optimize the use of the thrombin later in a narrow orificed dispenser, such as a sprayer, or expression through a thin tube. 
     Referring back to FIG. 1, attention is now directed to the clotting and adhesive protein processing unit  60 . All of the plasma not diverted to the thrombin processing unit  40  is admitted to an interior chamber  72  of the clotting and adhesive protein processing unit  60 . The clotting and adhesive protein processing unit  60  is manipulated by heat exchange and rotation so that all clotting and adhesive proteins extracted from the plasma will sediment at a nose  74  of the chamber  72  for subsequent extraction by means of a clotting and adhesive protein dispensing syringe  76  contained in a sterile pouch  78 . Chamber  72  is protected during this process by a filter vent  82  preventing contamination. Once the thrombin has been loaded into the dispensing syringe  42 , and the clotting and adhesive proteins have been loaded into the clotting and adhesive dispensing syringe  76 , the two syringes can be decoupled from the processing set  10  (e.g. sterile disconnect device), passed into the sterile, surgical arena where the contents are dispensed into sterile 3 cc plastic syringes which are subsequently loaded into the fibrin glue applicator for spraying or line and dot application. Mixing the thrombin with the clotting and adhesive proteins forms the biological glue. 
     Both dispensing syringes  42  and  76  are stored at room temperature, or preferably at 2  C. to 8  C. prior to usage. Please see FIGS. 13 through 16. 
     Assume 9-10 ml of room temperature plasma is introduced into the reaction chamber  26 . Add 1.0 ml of 75 mM calcium chloride (CaCl 2 ) and 2.0 ml of ethanol (ETOH) (i.e., ethanol taken from a 100% “stock” bottle and added to comprise 18.9% volume/unit volume or 15.02% ethanol weight/unit volume). The thrombin life A span is shown to have been at least 300 minutes while its clotting time is at 2.98 seconds. An ethanol final concentration range between 8.0% and 20.0% (volume/unit volume), however, still has utility. Please see FIG.  11 . 
     When the ethanol is at a final concentration of 18.9% volume/unit volume (as above) and the calcium chloride final concentration is 5.7 mM (1 ml taken from a 75 mM stock solution of calcium chloride), the thrombin lifespan also extends to at least 360 minutes while maintaining a clot time of 5.98 seconds. Calcium chloride final concentrations ranging between 4.5 mM and 23.0 mM, however, have utility. Please see FIG.  12 . 
     Solutions such as saline, dilute CaCl 2  (e.g. 125 mM CaCl 2 ) or even water added to the thrombin can alter both the clotting time and life span of the thrombin. Assume an ethanol final concentration of 18.9% and a calcium chloride concentration of 5.7 mM was used in the reaction chamber  26 . When the thrombin has been diluted 1 to 1.5 with water, the clot time has been extended to just less than 30 seconds, and has a life span of up to 150 minutes. 
     Moreover, having thus described the invention, it should be apparent that numerous structural modifications and adaptations may be resorted to without departing from the scope and fair meaning of the instant invention as set forth hereinabove and as described hereinbelow by the claims.