WO1991016063A1 - Systeme de cryoprecipitation de fibrinogene - Google Patents

Systeme de cryoprecipitation de fibrinogene Download PDF

Info

Publication number
WO1991016063A1
WO1991016063A1 PCT/US1991/002826 US9102826W WO9116063A1 WO 1991016063 A1 WO1991016063 A1 WO 1991016063A1 US 9102826 W US9102826 W US 9102826W WO 9116063 A1 WO9116063 A1 WO 9116063A1
Authority
WO
WIPO (PCT)
Prior art keywords
fibrinogen
plasma
freeze
freezing
recited
Prior art date
Application number
PCT/US1991/002826
Other languages
English (en)
Inventor
Shu T. Huang
Magnus Hook
Original Assignee
Uab Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Uab Research Foundation filed Critical Uab Research Foundation
Publication of WO1991016063A1 publication Critical patent/WO1991016063A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen

Definitions

  • the present invention relates to an improved method for preparing fibrinogen concentrate.
  • This invention also relates to the preparation of fibrinogen concentrate product which has clinical application as fibrin glue.
  • Sutures have been used as a conventional surgical means for uniting tissues and surgical margins, as hemostatic aids, and for blocking or ligation.
  • sutures suffer from many drawbacks.
  • sutures may be incompatible with the tissue, causing fistula or granuloma, sutures may cut through parenchymal and inflammatory tissues, absorbable suture material may disintegrate prematurely and produce dehiscence of the wound, and closely spaced sutures may cause tissue ischemia resulting in necrosis of the wound margins. Suturing is also time-consuming.
  • Cyanoacrylate-based substances have been commonly used as a fibrin glue. However, these substances are toxic to the tissue and cannot be absorbed (J.A. Collins et al., "Cyanoacrylate Adhesives as Topical Hemostatic Aids", Surgery
  • tissue glue was found to result in the growth of granulated tissue in response to the foreign substance, rejection of the cyanoacrylate, fistula formation and local suppuration.
  • fibrin powder could be utilized to achieve blood clotting and wound healing
  • Fibrin glue or sealant was successfully adapted for use in microvascular surgery. Others later combined suturing and sealing when applying the procedure in neurosurgery for extra-intracranial anastam ⁇ sis, and on the dura repair, satisfactory results were obtained using fibrin sealant.
  • Fibrin sealant has three components: fibrinogen concentrate, calcium chloride and thrombin. These components mimic the final common pathway of the clotting cascade, i.e. the conversion of fibrinogen to fibrin (see, e.g., R.W. Colman et al., Hemostasis & Thrombosis (2d ed.), 1987).
  • fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells.
  • Fibrinogen also has been found to stimulate fibroblast growth. The surface protein of fibroblasts has been found to contain fibronection.
  • thrombin of bovine origin is diluted with calcium chloride, with concentrations dependent on the tissue to be applied and the time of clotting. Equal amounts of fibrinogen concentrate and thrombin diluted in calcium chloride are used for clinical application. When the two components are mixed, thrombin converts fibrinogen to fibrin so that clotting is initiated and the mixture solidified. Meanwhile, in the presence of calcium ions, thrombin activates factor XIII to factor XllIa. Activated factor Xllla together with thrombin catalyzes the cross- linkage of fibrin and increases the strength of the clot. During wound healing the clot material undergoes gradual lysis and is completely absorbed.
  • fibrin sealant is used as a tool to facilitate hemostasis, permit tissue fixation, enhance implant material growth, stimulate fibroblast growth and as an embolization material.
  • Applications include orthopedic surgery, neural surgery, periodontal surgery, cerebral surgery, sinus or fistula obturation in proctologic and general surgery, chest surgery and genitourinary surgery, skin grafting in burn patients, punch hair grafting in plastic surgery, closure of corneal incisions in eye surgery, repair of lymph leak in general surgery and in myringoplasty in ear surgery.
  • fibrinogen is prepared from the plasma pooling of a large numbers of donors, which has high potential for disease transmission. In addition, fibrinogen will not tolerate the ten hours of heating at 60°C used to inactivate the hepatitis virus in other blood fractions. Studies have indicated that this product was a source of hepatitis transmission (7.8% of post-transfusion hepatitis rate) . Under these circumstances, the FDA revoked all licenses for the manufacture of human fibrinogen since June 30, 1978.
  • fibrinogen product is commercially available as a fibrinogen concentrate kit ("Tisseel", Immuno AG, Vienna, Austria) prepared from pooled fresh frozen plasma.
  • the tensile strength for Tisseel is 900 g/cm 2. Since this commercial fibrinogen concentrate is not available in the United States because it is currently not licensed by the FDA, alternative methods such as chemical precipitation and cryoprecipitation have been used to prepare fibrinogen concentrates.
  • Fibrinogen is one of the three main protein constituents of plasma.
  • the major constituent, albumin (ALB) occurs in a concentration of approximately four per cent.
  • the plasma globulins are present in a concentration of about 2.5 per cent and are particularly associated with the processes of immunity. Fibrinogen occurs in much smaller amounts, with its concentration in human plasma being about 0.4 per cent.
  • the plasma proteins can be separately isolated by: 1) organic solvents such as methanol or ethanol at low temperature using Cohn's fractionation, 2) cryoprecipitation, 3) chemical precipitation of plasma with salts such as ammonium sulfate, potassium phosphate, and sodium citrate, and 4) other methods.
  • organic solvents such as methanol or ethanol at low temperature using Cohn's fractionation
  • cryoprecipitation 3) chemical precipitation of plasma with salts such as ammonium sulfate, potassium phosphate, and sodium citrate
  • salts such as ammonium sulfate, potassium phosphate, and sodium citrate
  • the solubility of the plasma proteins in these substances decreases in the order of albumin, globulin, and fibrinogen. The latter precipitates first and albumin last upon the addition of increasing amounts of the precipitating agent.
  • Fibrinogen is the first material precipitated and harvested at -5°C with 25% ethanol at a pH of 6.9.
  • Variables determining the precipitation of proteins are ethanol concentration, pH, temperature, ionic strength and protein concentration.
  • the standard cryoprecipitation method has been primarily used to prepare antihemophilic factor (Factor VIII).
  • Cryoprecipitate also has been known as a source of fibrinogen.
  • the cryoprecipitate method can be also used to prepare fibrinogen concentrate. It is known that some factors might affect the yield of Factor VIII, such as ABO blood grouping, freezing and thawing conditions (see Kasper et al., "Determinants of Factor VIII Recovery in Cryoprecipitate", Transfusion 15, 312-322, 1975, and Rock et al., "Variations in Cryoprecipitate Production", Transfusion 17, 50-53, 1977).
  • Human fibrinogen can be precipitated from human plasma by ammonium sulfate, polyethylene glycol, polyvinyl-pyrrolidone, and barium/magnesium sulfate. Entering the closed blood bag system for the addition of chemicals opens the system to the potential for bacterial contamination. Small amounts of fibrinogen concentrate solution (0.5 - 1.9 ml) can be prepared using these methods, but the side effects and safety due to the chemical substances as well as bacterial contamination opportunities are of great concern.
  • cryoprecipitation method is the simplest and most economic way to make concentrated fibrinogen.
  • Most U.S. blood banks use cryoprecipitate as the fibrinogen (FBG) source for fibrin glue which contains less FBG (260-2,500 mg/dl) compared to Tisseel (7,000-10, 000 mg/dl).
  • Fibrinogen concentrate can be prepared from random singledonor fresh frozen plasma or autologous plasma in sufficient quantity to meet some surgical demand. According to the Standards of the American Association of Blood Banks, fibrinogen concentrate can be currently stored for up to 5 years at -80°C or at least 5 days at 4°C until it is needed. Cryoprecipitate contains Factor VIII and fibrinogen and is used to supply fibrinogen in patients with hypofibrinogemia and also as an alternative source of fibrinogen concentrate for fibrin sealant in the United States.
  • An object of this invention is to achieve a method of isolating fibrinogen which can be carried out in blood banks, which follows the Standards of American Association of Blood Banks for preparation conditions in a closed bag system, and which also produces a high yield of fibrinogen capable of producing a clot of high tensile strength.
  • An additional object is to produce a fibrinogen concentrate for use in fibrin glue and the like, which has increased fibrinogen concentration and increased tensile strength.
  • Another object is to obtain a fibrinogen concentrate according to a method which overcomes the disadvantages of the known methods.
  • fibrinogen concentrate is prepared by a cryoprecipitation method which employs at least two freeze-thaw cycles.
  • a cryoprecipitation method according to the present invention concentrates fibrinogen by the steps of: separating plasma from whole blood; subjecting the plasma to a first freeze-thaw cycle by freezing the plasma at a temperature of about -18°C or below (e.g., at least -32°C), and then thawing the frozen plasma at a temperature of about 4°C; subjecting the plasma to a second freeze-thaw cycle by freezing the plasma at a temperature of about -18°C or below, and then thawing the frozen plasma at about 4°C; centrifuging the plasma to concentrate cryoprecipitated fibrinogen; and separating the fibrinogen from the plasma.
  • the method may be desirably limited to only these steps.
  • freeze-thaw cycles include slow-freezing and quick-thawing (for about 2-3 hours).
  • a fibrinogen product prepared in accordance with this invention advantageously has a higher concentration and increased volume of fibrinogen (about 4,000-6,000 mg/dl FBG in 3-5 ml) than achieved by conventional cryoprecipitation methods.
  • a fibrinogen product can be readily prepared having from about 2-6 times more fibrinogen than the standard cryoprecipitation method.
  • the fibrinogen concentrate prepared according to the invention can be used to make an improved fibrin glue.
  • the fibrinogen concentrate can be made from random single-donor fresh frozen plasma or autologous plasma prior to elective surgery.
  • Fig. l represents a correlation of thrombin clot assay and heat precipitation method to determine fibrinogen concentration.
  • Fig. 2 shows 3.5% polyacrylamide gel electrophoresis results.
  • A is human serum, diluted 1:2;
  • B is human plasma, diluted 1:2;
  • C is fibrinogen concentrate prepared by freezethaw one time method, diluted 1:10;
  • D is fibrinogen concentrate prepared by freeze-thaw two times method, diluted 1:10, and;
  • E is pooled fibrinogen concentrate, diluted 1:10.
  • Fig. 3 gives the results of SDS-polyacrylamide gel electrophoresis on cryoprecipitates and supernatants.
  • A shows molecular weight markers, high and low; B is standard cryoprecipitate, diluted 1:10; C is standard cryoprecipitate supernatant, diluted 1:10; D is citrated plasma, diluted 1:10, and; E, F and G are supernatant after prepared fibrinogen concentrate, diluted 1:10.
  • Fig. 4 contains the results of SDS-polyacrylamide gel electrophoresis under reduced conditions of fibrinogen concentrates.
  • A represents molecular weight markers, high and low;
  • B is fibrinogen standard, diluted 1:50;
  • C is fibrinogen concentrate prepared by the freeze-thaw one time method, diluted 1:50;
  • D is fibrinogen concentrate prepared by the freeze-thaw two times method, diluted 1:50, and;
  • E and F are pooled fibrinogen concentrate, diluted 1:50.
  • Fig. 5 represents SDS-polyacrylamide gel electrophoresis under non-reduced conditions of fibrinogen concentrates, wherein A-F have the same meaning as in Fig. 4.
  • FIG. 6 shows the results of agarose gel electrophoresis.
  • A represents human serum; B is human plasma; C is fibrinogen concentrate prepared by freeze-thaw one time method, diluted 1:10; D is fibrinogen concentrate prepared by freeze-thaw two times method, diluted 1:10; E and F are pooled fibrinogen concentrate, diluted 1:10; G is fibrinogen standard, diluted 1:10, and; H is human plasma.
  • Fig. 7 is a schematic representation of the fibrinogen attachment study, showing the processing of cryoprecipitate and addition of PFBG. Detailed Description of the Invention
  • the new method includes double freeze-thaw cycles on plasma, centrifugation at 4200 rpm for 12 minutes (Beckman J-6B centrifuge), and draining of the plasma to obtain fibrinogen concentrate.
  • the preferred conditions of our improved method to prepare fibrinogen concentrate are freezing slowly and thawing quickly (4°C water bath, 2-3 hrs.). More particularly, the freezing steps in the F/T cycles involve freezing in a liquid immersion type freezer (e.g., "INSTACOOL" freezer), and the thawing steps utilize a 4°C circulating water bath.
  • INSTACOOL liquid immersion type freezer
  • whole blood 450-500 ml
  • centrifuged at 4200 rpm 4200 rpm
  • the plasma is separated into an attached bag.
  • plasma (210-300 ml) is frozen at less than -32°C in a liquid immersion freezer
  • the plasma is next subjected to a second freeze-thaw cycle.
  • the plasma is refrozen at less than -18°C in the immersion freezer and stored for at least 18 hours. Then the plasma is rethawed in a 4°C circulating water bath for 2 hours.
  • the plasma is centrifuged at 4200 rpm (Beckman JB6) for 12 minutes at 0°C to concentrate the cryoprecipitated fibrinogen.
  • the bag of plasma is inverted and the supernatant plasma drained off to leave the fibrinogen concentrate (3-5 ml).
  • the fibrinogen concentration can be at least doubled, the fibrinogen recovery rate can be increased by 2.5-fold, clottable protein can be increased by 1.5-fold, tensile strength can be improved by 2-5-fold and the volume can be increased.
  • a fibrin sealant prepared from cryoprecipitated fibrinogen concentrate according to the invention can include an amount of fibrinogen concentrate equal to the amount of thrombin.
  • Fresh frozen plasma in citrate phosphate dextrose anticoagulant (Fenwal Laboratories, Deerfield, IL) was obtained from American Red Cross, Alabama Region, and stored at
  • fibrinogen concentrate for fibrin glue
  • total protein and fibrinogen quantitative and qualitative concentrations were determined.
  • fibrinogen concentrate was tested by polyacrylamide gel electrophoresis and agarose gel electrophoresis for purity and to determine the mechanism for increased yield. Fibrinogen attachment and conversion tests were used to study the mechanism of increasing fibrinogen yield in our improved method. Sacrificed rat skin was tested with different concentrations of fibrinogen to determine the binding strength of the fibrin glue when clinically applied.
  • a cryoprecipitation method was used throughout experimentation. Thirty units of group O fresh frozen plasma were used to determine if repeating the freezing and thawing procedure would produce different yields of fibrinogen concentrate.
  • the first ten units of fresh frozen plasma stored at ⁇ - 18°C were removed from a freezer (Forma Scientific, Mallinckrodt Inc., Marietta, OH) and put in a 4°C walk-in refrigerator (Forma Scientific, Mallinckrodt Inc., Marietta, OH) in a Blood Bank for at least 12 hours to thaw completely. After the plasma was thawed completely, the bag was placed in the 4°C refrigerated centrifuge (Sorvall RC-3, Newtown, Conn.), and centrifugated at 2,750-3,500 X g for ten minutes to bring down the cryoprecipitate to the bottom of the bag.
  • 4°C refrigerated centrifuge Sorvall RC-3, Newtown, Conn.
  • the bag was inverted with the port portion of the bag at the bottom and then all the plasma was drained by gravity into another transfer bag. The tubing was heat sealed and the bags were separated. The original bag contained fibrinogen concentrate.
  • the bag was placed in a 37°C water bath (blood thawing bath model 2031, Forma Scientific, Mallinckrodt Inc., Marietta, OH) for 10 minutes and the contents were aspirated into a syringe using a medical injection site via bag port. This is the so called standard procedure, or freeze-thaw one time method.
  • the second ten units of fresh frozen plasma were removed from the ⁇ -18°C freezer and thawed under the same conditions as above. After the plasma was completely thawed, the bags were placed back into ⁇ -18°C freezer (Kelvinator Commercial Products, Inc., Manitowoc, Wis.) again. The plasma bags were kept in the freezer at least 12 hours to freeze solidly. The plasma was removed from the freezer again, and processed the same as the first ten units for further testing. This procedure in accordance with our invention is termed the freeze-thaw two times method. The third ten units of fresh frozen plasma were removed from the freezer and thawed under the same conditions as above, except the freezing and thawing procedure was carried out three times. After the last thawing, these units were processed as the first ten units of plasma. This procedure is called the freeze-thaw three times method.
  • thawing condition on fibrinogen yield Sixty- five units of fresh frozen plasma were used to study the effect of thawing conditions using a 4°C refrigerator (Forma Scientific, Mallinckrodt Inc., Marietta, OH) or 4°C circulating water bath (model 2386 cryoprecipitate bath. Forma Scientific, Mallinckrodt Inc., Marietta, OH) on the harvest of fibrinogen. Twenty-three units were processed by the freeze-thaw one time method. The remaining 42 units were processed by the freeze-thaw two times method. Each group included both thawing conditions.
  • the effect of mixing on fibrinogen yield Eight units of fresh frozen plasma were used to study the effect of mixing or non-mixing during processing on the fibrinogen yield. The freeze-thaw two times and three times procedures were investigated. The mixing groups of plasma bags were mixed by inverting the bags several times every 30 minutes during thawing. The non-mixing groups of plasma bags were placed in the refrigerator without being disturbed until completely thawed.
  • the effect of centrifugation on fibrinogen yield To study the effect of centrifugation between the freeze and thaw procedure, twenty units of fresh frozen plasma were pooled and studied. The pooled plasma was divided into 18 transfer bags with 250 ml plasma in each with an equal number of bags included in the two sets. Set I was processed by the standard freeze-thaw one time procedure. Set II was processed by the freeze-thaw two times procedure, but the plasma bag was centrifuged at 3,000 x g for 10 minutes to bring down the precipitate after first thawing, then processed as previously described.
  • the effect of freezing time on fibrinogen yield Another eighteen units of fresh frozen plasma were included in the freezing time study. Two different kinds of freezers were used: the FORMA Blast freezer (model 8114, Forma Scientific, Mallinckrodt Inc., Marietta, OH) and the INSTACOOL freezer (InstaCool, North America, Anaheim, CA). The Instacool freezes relatively slowly and the former cold blast freezer freezes faster. All units were processed by the freeze-thaw two times procedure. The fibrinogen concentrations of the final products were compared.
  • ABO blood grouping The effect of ABO blood grouping on fibrinogen yield: To determine whether ABO grouping would affect the fibrinogen yield when using different ABO blood group plasmas, 51 units of different ABO group plasma were investigated. These included 16 units of group A, 5 units of group B, 28 units of group 0, and 2 units of group AB plasma. The freeze-thaw two times procedure was used.
  • the Lowry (Folin-Ciocalteu) method was used to determine the total protein content of fibrinogen concentrates (Lowry et al., "Protein Measurement with the Folin Phenol Reagent", J Biol Chem 193, 265-275, 1951).
  • Purified human fibrinogen (Sigma Chemical Company, St. Louis, MO) was used as a standard. The standard was diluted to 0.5 mg/ml using distilled water, and several different amounts of this diluted solution were used to set up the calibration curve. Samples were diluted 1:250 using distilled water for testing.
  • reagent A prepared by mixing sodium tartrate, copper sulfate and sodium carbonate as described by Lowry (Horowitz et al., "Preparation of Antihemophilic Factor from Human Plasma Cryoprecipitate", Transfusion 24, 357-362, 1984)
  • reagent B diluted Folin reagent (I N) as described by Lowry
  • the optical density (OD) of each reaction mixture was determined at 500 nm against a distilled water blank. The average OD of the reagent blanks was subtracted from the average OD of each duplicate standard and sample.
  • the calibration curve was plotted with OD as the ordinate and protein concentration as the abscissa. Using the average OD obtained for the sample, the protein concentration was read directly from the calibration curve. If the ODs of the testing samples were over 3.5 (outside the calibration curve maximum), these samples were then serially diluted until they could be calculated from the calibration curve.
  • Purified human fibrinogen (Sigma Chemical Company, St. Louis, MO) was used as the standard throughout the tests.
  • concentration of fibrinogen in the standard, the fibrinogen concentrate (for fibrin glue) and the original fresh frozen plasma from the blood bag segment were tested by Thrombin Clot Opacity method and the Heat Precipitation method (Foster et al., "Determination of Plasma Fibrinogen by Means of Centrifugation After Heating", Am J Clin Path 31, 42-45, 1959). All test samples were diluted 1:11 with distilled water.
  • Thrombin Clot Opacity Method to determine the fibrinogen concentration was performed using ACATM Du Pont Discrete Clinical Analyzer (E.I. Du pont de Nemours & Co. Inc., Wilmington, DE). The principle of the procedure is as follows: human thrombin catalyzes the conversion of fibrinogen to insoluble fibrin polymer. The rate of this reaction is enhanced in the presence of dextran and calcium chloride, while polybrene neutralizes heparin up to 10 USP units/ml which might be present in the patient's plasma.
  • the rate of increase of absorbance at 340 nm due to the increase in fibrin polymerization is related to the functional fibrinogen concentration by means of a standard curve, or a mathematical function.
  • Fibrinogen recovery percentage was calculated by the following equation:
  • sample buffer 5% tris-glycerin buffer stock solution, pH 8.9
  • sample buffer 5% tris-glycerin buffer stock solution, pH 8.9
  • Finished gels were fixed with fixing solution (57.0 gm trichloroacetic acid, 17.0g sulfosalicylic acid, 150 ml methanol in 500 ml of distilled water) for about 60 minutes, then fast stained with 0.1% Coomassie Blue (Sigma, St. Louis, MO) at room temperature for 30 minutes and then diffusion-destained by repeated washing in 7% acetic acid.
  • the destained gels were soaked in preserving solution (300 ml ethanol, 100 ml acetic acid, and 100 ml glycerol in 1 liter of distilled water) for 2 hours and dried on a gel bound film.
  • Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to evaluate the purity and to separate the polypeptide chain of the different fibrinogen products.
  • Polyacrylamide gradient gel a mixture of 3-10%, was prepared.
  • Hoefer electrophoresis system (Model SE 690, serial 0018, Hoefer Scientific Instruments, San Francisco, CA) was used to carry out the whole electrophoresis procedure.
  • Hoefer gradient mixer SG series, Hoefer Scientific Instruments, San Francisco, CA
  • P-S peristaltic pump Pharmacia, Laboratory Separation Division, Uppsala, Sweden
  • Tris-SDS buffer contain dithioerythritol (DTE) and bromphenol blue loading dye, and were placed in 100°C heat block and boiled for 5 minutes. Unreduced samples were prepared with tris-sodium dodecyl sulfate (Tris-SDS) buffer and dye but were not boiled. Samples were stacked with gel buffer on a gel apparatus and were tested at 400 V for 80 minutes. Finished gels were fixed with fixing solution for 30 minutes first, then fast stained with Coomassie blue and destained.
  • DTE dithioerythritol
  • Tris-SDS tris-sodium dodecyl sulfate
  • CORNING Electrophoresis System (Corning Medical, Palo Alto, CA) was used to perform the agarose gel electrophoresis.
  • the gel film consisted of 1% (w/v) agarose, 5% (w/v) sucrose, and 0.035% (w/v) ethylenediaminotetraacetate (EDTA) disodium salt in a 0.065 M barbital buffer.
  • the gel contained eight wells and each well was filled with 1 ul of diluted sample.
  • the gel was electrophoresad for 50 minutes and then reduced to a thin film by pressing. After pressing, the eel was dried to a fine film using hot air from a hair dryer.
  • the gel was then placed in staining solution (1,25 g amido black 10 B dissolved in 250 ml destaining solution) for 5 minutes. Excess stain was removed by soaking the gel in destaining solution (1710 ml ethanol, 1710 ml distilled water and 380 ml acetic acid) and changing the solution several times. Once again the gel was dried with a stream of hot air.
  • another three groups (n-4 each) were also included in the study.
  • OPD o-phenylenediamine
  • Tc measure the tensile strength
  • highly concentrated human fibrinogen obtained from pooled human plasma
  • distilled water was diluted with distilled water into the following concentrations: 2,000 mg/dl, 4,000 mg/dl and 6,000 mg/dl.
  • Thrombin 1,000 IU was diluted with 40 mmol of calcium chloride solution to 500 IU/ml.
  • Diluted fibrinogen concentrate and thrombin solution were filled into 2 separate syringes until ready for use.
  • Two 2 cm X 2 cm split-thickness rat skin epidermis sides were glued onto a measuring device with cyanoacrylate glue (Super glue Corporation, Hollis, NY).
  • autologous fibrinogen concentrate was prepared according to our improved technique and used as fibrin glue to apply skin grafts on patients with facial burns. Burns that were not essentially healed at 10 days were excised and grafted with non-meshed split-thickness skin grafts secured with autologous fibrin glue.
  • the results of determining the effect of thawing condition on fibrinogen yield are shown in Table 5.
  • the units thawed in the refrigerator had increased volume but lower fibrinogen concentration.
  • the units thawed in the circulating water bath had decreased volume but higher fibrinogen concentration.
  • the total fibrinogen amounts were about the same.
  • the freeze-thaw two times method was the most advantageous method to prepare fibrinogen concentrate for fibrin glue.
  • Plasma thawed, in a 4°C refrigerator or 4°C circulating water bath had the same amount of fibrinogen yield. There was no need for mixing the plasma while thawing. Centrifugation between freeze-thaw cycles had no measured benefit. Longer freezing times but shorter thawing times resulted in better fibrinogen yields and product volumes. ABO blood grouping was not an important factor for preparing high yield fibrinogen concentrates.
  • the proposed mechanism for the increased fibrinogen yield is that residual fibrinogen in the supernatant attaches to the previously cryoprecipitated protein. This attached fibrinogen is incorporated into the cryoprecipitated protein during the second freezing and thawing which then generates the attachment of more fibrinogen.
  • Fibrinogen concentrates prepared by the standard method had an average 50% clottable protein which was significantly less than 74.3% for freeze-thaw two times and 74.9% for freeze- thaw three times method (p ⁇ 0.005). See Tables 10-13.
  • Figure 7 shows a diagram of the three groups in this study and when peroxidase-labeled fibrinogen (PFBG) was added.
  • Table 17 shows the fibrinogen concentrations before and after the experiment. This correlated with the finding that freeze-thaw two or three times had more fibrinogen precipitated out.
  • Table 18 shows the EIA results of the attachment study and that in groups IA, IIA, IIIA the more the freeze-thaw cycle was repeated, the higher the optic density of the product mixture. This appears to indicate that fibrinogen attaches to cryoprecipitated protein.
  • the tensile strength of the fibrinogen sealant is related to the concentration of fibrinogen in this product.
  • the tensile strength was dramatically increased when the fibrinogen concentration was higher than 4,000 mg/dl.
  • it is very important to have a highly concentrated fibrinogen product for clinical application to ensure sufficient tensile strength for stopping bleeding quickly.
  • the method according to the invention may advantageously be limited to of the steps of separating plasma from whole blood, subjecting the plasma to two freeze-thaw cycles, centrifuging the plasma and then separating the fibrinogen product from the plasma.
  • ABO grouping is a concern when making fibrinogen concentrate because of the association of decreased Factor VIII found in the cryoprecipitate of group O donors. According to our data (Table 9), ABO blood grouping was not an important factor in obtaining high yield fibrinogen concentrates.
  • centrifugation between the freeze-thaw cycle would increase the fibrinogen harvest, since centrifugation should bring down the cryoprecipitate that was already present in the plasma and then the supernatant plasma would form more cryoprecipitate.
  • centrifugation between the freezing and thawing procedure had no benefit as demonstrated by our results. Centrifugation packed the precipitate into a small pellet reducing the precipitate surface dramatically. Under these circumstances only a small portion of the suspended plasma fibrinogen could be attracted onto the precipitation, resulting in only a little or even no increase in the fibrinogen yield.
  • fibrinogen molecules polymerized into a bigger macromolecule. After the first freezing and thawing, more fibrinogen would attach to the fibrinogen macromolecules. Then these molecules would become larger. The 3.5% polyacrylamide gel electrophoresis and agarose gel electrophoresis failed to show this.
  • the quantity of PFBG attached to the cryoprecipitated protein was greater than the PFBG directly converted to cryoprecipitate. This ratio was up to 5:1 (see Table 18, group IB and IIA). This showed that the effect of fibrinogen attachment onto the cryoprecipitated protein might be more important than the fibrinogen directly converted into cryoprecipitate. After the repeated freezing and thawing (IIB and IIIA), the attachment effect (IIIA) was again more prominent than fibrinogen conversion (IIB). The fibrinogen attached to the cryoprecipitated protein in the first thawing of the plasma was incorporated into the larger cryoprecipitate mass during the second freeze-thaw cycle, which then attracted more fibrinogen attachment to the cryoprecipitate.
  • the supernatant subsequently formed additional small amounts of cryoprecipitated protein which again enhanced more free PFBG to attach. This may explain why repeated freezing and thawing yielded 2-3 fold greater amounts of fibrinogen concentrate than the freeze- thaw one time method. A limited increase in PFBG attachment was noted in the third freezing and thawing. This may be due to depletion of residual fibrinogen in the supernatant.
  • Fibrinogen concentrates prepared by our improved method were used clinically to improve the early adherence of skin grafts to difficult wounds that would otherwise require bolster dressing and long-term immobilization.
  • a study in 22 patients utilizing autologous fibrin glue for facial burns in accordance to the aesthetics units and burns in selected difficult sites was undertaken. All grafts demonstrated excellent survival without suturing or pressure dressings, allowing early mobilization, ambulation and minimal post-op care. Color match and texture were excellent after 14 months.
  • single-random units of plasma may be used so that the fibrinogen concentrate will have a low risk for blood transmitted diseases.
  • the invention is used with autologous plasma there is no such risk.
  • a frozen stock of fibrinogen concentrate can be kept at -80°C for up to five years which meets the Standards of the American Association of Blood Banks. The method is simple and can be easily performed in any blood bank laboratory that has a refrigerated centrifuge.
  • the high quality and quantity of the fibrinogen concentrate of the invention will meet the clinical need for preparing fibrin sealant.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Procédé de cryoprécipitation permettant de concentrer le fibrinogène et produit de fibrinogène ainsi préparé, consistant à séparer le plasma du sang total, le plasma étant soumis à deux cycles de congélation-décongélation dans un système de sac fermé, le plasma étant ensuite centrifugé et le produit de fibrinogène concentré étant séparé du plasma. Dans les cycles de congélation-décongélation, le plasma est de préférence congelé lentement dans un congélateur à immersion à une température d'environ moins 10 °C ou inférieure, et décongelée rapidement à une température d'environ 4 °C. Le procédé augmente le rendement en produit ainsi que la concentration du fibrinogène. On améliore la résistance à l'attraction du concentré de fibrinogène. On peut utiliser le produit de fibrinogène afin de préparer de la colle fibrineuse à partir d'un seul donneur aléatoire ou de plasma autologue. Le concentré de fibrinogène a une concentration d'environ 2000 à 6000 mg/dl ainsi qu'un volume compris entre environ 3 et 5 ml.
PCT/US1991/002826 1990-04-26 1991-04-25 Systeme de cryoprecipitation de fibrinogene WO1991016063A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US51551090A 1990-04-26 1990-04-26
US515,510 1990-04-26

Publications (1)

Publication Number Publication Date
WO1991016063A1 true WO1991016063A1 (fr) 1991-10-31

Family

ID=24051661

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/002826 WO1991016063A1 (fr) 1990-04-26 1991-04-25 Systeme de cryoprecipitation de fibrinogene

Country Status (1)

Country Link
WO (1) WO1991016063A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011925A1 (fr) * 1996-09-18 1998-03-26 Flavio Tarantino Procede de preparation d'une colle de fibrine autologue
US6110484A (en) * 1998-11-24 2000-08-29 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
WO2004039382A1 (fr) * 2002-10-25 2004-05-13 Shanbrom Technologies, Llc Production amelioree de facteurs de coagulation par cryoprecipitation
US9358318B2 (en) 2004-10-20 2016-06-07 Ethicon, Inc. Method of making a reinforced absorbable multilayered hemostatic wound dressing
US9439997B2 (en) 2004-10-20 2016-09-13 Ethicon, Inc. Reinforced absorbable multilayered hemostatis wound dressing
CN112146955A (zh) * 2020-09-23 2020-12-29 深圳市亚辉龙生物科技股份有限公司 血清的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3920625A (en) * 1973-06-19 1975-11-18 Kabi Ab Isolation of coagulation factors from biological material using cross linked sulfated, sulfonated carbohydrates
US4627879A (en) * 1984-09-07 1986-12-09 The Trustees Of Columbia University In The City Of New York Fibrin adhesive prepared as a concentrate from single donor fresh frozen plasma

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3920625A (en) * 1973-06-19 1975-11-18 Kabi Ab Isolation of coagulation factors from biological material using cross linked sulfated, sulfonated carbohydrates
US4627879A (en) * 1984-09-07 1986-12-09 The Trustees Of Columbia University In The City Of New York Fibrin adhesive prepared as a concentrate from single donor fresh frozen plasma

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BLUT, Vol. 56, No. 3, March 1988, SMIT SIBINGA et al., "Double Cryoprecipitated Factor VIII Concentrate from Heparinised Plasma and its Heat Treatment", pages 111-116. *
J. CLIN. PATHOL., Vol. 38, 1985, FARRUGIA et al., "Studies on the Procurement of Blood Coagulation Factor VIII: Effects of Plasma Freezing Rate and Storage Conditions on Cryoprecipitate Quality", pages 433-37. *
JAPAN. J. EXP. MED., Vol. 39, No. 2, 1969, SATO et al., "Preparation of Plasminogen-Free Fibrinogen", pages 135-42. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011925A1 (fr) * 1996-09-18 1998-03-26 Flavio Tarantino Procede de preparation d'une colle de fibrine autologue
US6110484A (en) * 1998-11-24 2000-08-29 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
US6277394B1 (en) 1998-11-24 2001-08-21 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
WO2004039382A1 (fr) * 2002-10-25 2004-05-13 Shanbrom Technologies, Llc Production amelioree de facteurs de coagulation par cryoprecipitation
US9358318B2 (en) 2004-10-20 2016-06-07 Ethicon, Inc. Method of making a reinforced absorbable multilayered hemostatic wound dressing
US9439997B2 (en) 2004-10-20 2016-09-13 Ethicon, Inc. Reinforced absorbable multilayered hemostatis wound dressing
CN112146955A (zh) * 2020-09-23 2020-12-29 深圳市亚辉龙生物科技股份有限公司 血清的制备方法

Similar Documents

Publication Publication Date Title
US4377572A (en) Tissue adhesive
US4414976A (en) Tissue adhesive
US5330974A (en) Therapeutic fibrinogen compositions
Brennan Fibrin glue
Weksler et al. Platelet-dependent generation of chemotactic activity in serum
JP2987207B2 (ja) 血小板因子に富む生物接着剤の調製方法及びその適用
Becker et al. Contractile proteins of endothelial cells, platelets and smooth muscle
US4627879A (en) Fibrin adhesive prepared as a concentrate from single donor fresh frozen plasma
US4928603A (en) Method of preparing a cryoprecipitated suspension and use thereof
AU710720B2 (en) Platelet glue wound sealant
US5792835A (en) Method of preparing a topical fibrinogen complex
WO1992013495A1 (fr) Adhesif a base de fibrinogene
Fattahi et al. Clinical applications of fibrin sealants
JPS5826821A (ja) 組織接着剤並びにその製造方法
Altmeppen et al. Composition and characteristics of an autologous thrombocyte gel
Kumar et al. Stability of human thrombin produced from 11 ml of plasma using the thrombin processing device
Plow et al. Fibronectin binding to thrombin-stimulated platelets: evidence for fibrin (ogen) independent and dependent pathways
WO1991016063A1 (fr) Systeme de cryoprecipitation de fibrinogene
US4347243A (en) Acid soluble, pepsin resistant platelet aggregating material
US6916911B1 (en) Use of fibrinogen multimers
JPH0199565A (ja) フィブリン糊調製用キット
Silberman et al. Effects of ancrod (Arvin) in mice: studies of plasma fibrinogen and fibrinolytic activity
Ubezio et al. Bio-modulators in platelet-rich plasma: a comparison of the amounts in products from healthy donors and patients produced with three different techniques
US4423036A (en) Acid soluble platelet aggregating material isolated from human umbilical cord
Takahashi et al. Interaction of collagen with serum complement: inhibition of complement-mediated hemolysis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE