CN1113656C - Precipitation of growth-factor-enriched fibrinogen concentrate from platelet rich plasma - Google Patents

Precipitation of growth-factor-enriched fibrinogen concentrate from platelet rich plasma Download PDF

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Publication number
CN1113656C
CN1113656C CN98810252A CN98810252A CN1113656C CN 1113656 C CN1113656 C CN 1113656C CN 98810252 A CN98810252 A CN 98810252A CN 98810252 A CN98810252 A CN 98810252A CN 1113656 C CN1113656 C CN 1113656C
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China
Prior art keywords
rich
blood plasma
hematoblastic
centrifugal
hematoblastic blood
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Expired - Fee Related
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CN98810252A
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CN1276725A (en
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卢·布拉塞蒂
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Harvest Technologies Corp
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Harvest Technologies Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028—Polypeptides; Proteins; Degradation products thereof
    • A61L26/0042—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
    • B01D17/02—Separation of non-miscible liquids
    • B01D17/0217—Separation of non-miscible liquids by centrifugal force
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
    • B01D17/02—Separation of non-miscible liquids
    • B01D17/04—Breaking emulsions
    • B01D17/047—Breaking emulsions with separation aids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/01—Separation of suspended solid particles from liquids by sedimentation using flocculating agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
    • B01D21/262—Separation of sediment aided by centrifugal force or centripetal force by using a centrifuge
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/30—Control equipment
    • B01D21/34—Controlling the feed distribution; Controlling the liquid level ; Control of process parameters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2221/00—Applications of separation devices
    • B01D2221/10—Separation devices for use in medical, pharmaceutical or laboratory applications, e.g. separating amalgam from dental treatment residues

Abstract

Increased fibrinogen yields are obtained by adding a precipitating agent to plasma having a high platelet concentration, such as platelet rich plasma. The precipitating agent may be any of several known agents, including polyethylene glycol and ammonium sulfate. The platelet rich plasma is obtained in the preferred embodiment by subjecting plasma to 'soft spin' centrifugation of about 580G. An automatic, multiple decanting and multiple-speed centrifuge is preferably used to separate anti-coagulated whole blood into the platelet rich plasma component and red blood cells. The proteins, preferably fibrinogen, FXIII, and FVIII, in the platelet rich component are precipitated, and the proteins and platelets are then concentrated by further centrifugation.

Description

The fibrinogen concentrate of precipitation of growth-factor-enriched from be rich in hematoblastic blood plasma
Technical field
The application relates to improving one's methods of collection and deshydremia component.Particularly, the present invention relates to prepare growth-factor-enriched fibrinogen concentrate from being rich in hematoblastic blood plasma.
Background technology
People look forward to a kind of method that can concentrate and collect some hematoglobin protein in the system of sealing apace from whole blood always, described hematoglobin protein also comprises platelet and some somatomedin, so that help the doctor to be used to make wound closure, stop blooding, prevent air and fluid seepage quickly and help quickly-healing and carry out medicine and biological the transmission.
Skilled in the art will recognize that when collect on one's body from the patient at intra-operative be rich in hematoblastic blood plasma after and it is mixed with thrombin (usually in 7: 1 ratio), place them in the wound then, in after this several seconds, just have platelet gel and form.This gel can both reach the hemostatic effect quickly than the hemorrhage of other any routine.Because this gel has heavy-gravity character, it can also block air and fluid seepage, because the existence of platelet derived growth factor (PDGF) finally causes wound to heal quickly.This gel only contains Fibrinogen, FXIII, FVIII and the PDGF of natural horizontal.Therefore, lower than desired usually by the formed adhesion of this gel, tension force and shearing force.Because the content of desirable proteins is low, even also can produce the thing of hemostasis or shutoff failure, cause to realize desired result.
Collect in patient's body during corrective surgery and be rich in hematoblastic blood plasma and need one " blood processor ", it is to sell under the name of " Cell Saver " trade mark that a kind of blood processor is arranged, and other also is known by this type of device that each company makes." Cell Saver " needs one high degree of skill is arranged, and sometimes or even titular operator, opens and operate this device.Need heavy caliber vein or tremulous pulse interface in the operating process (generally continuing 30-60 minute) and need to handle the blood of several liters of as many as to obtain and to collect the platelet and the blood plasma of enough volumes.Patient's blood flow state and heart state must be very stable, could allow to handle so a large amount of blood.
United States Patent (USP) 5707331 (people such as Wells.) has been described and has been used to obtain autologous fibrinogenic automated system, and the content of this patent disclosure is incorporated herein does reference.According to this system, the whole blood of relatively small amount (for example 50ml) is put into first Room of the detachable container in 2-chamber.Second Room is put in the fibrinogen deposition agent.Then container is placed centrifuge, and centrifugal to whole blood to isolate blood plasma, produce the blood plasma of platelet poorness.In blood plasma impouring second Room with the platelet poorness that so obtains, mix with precipitant (for example PEG or ammonium sulfate) at that.And then centrifugal to blood plasma and precipitant, be used for and the blended fibrin precipitation of thrombin with acquisition, prepare fibrin sealant.
The description of preferred embodiment
Collecting a key factor of fibrinogenic method, as be described in the above-mentioned United States Patent (USP) 5707331 those, is to be collected in the percentage ratio that Fibrinogen in the precipitation accounts for whole blood.The applicant finds, when when wherein being settled out fibrinogenic blood plasma and containing the platelet of higher level, and above-mentioned important factor, promptly " fibrinogenic productive rate " is just unexpectedly higher.Therefore, the method according to this invention joins known fibrinogen deposition agent and is rich in the hematoblastic blood plasma, can obtain the Fibrinogen of high yield.
It is about 50% that the fibrinogenic productive rate that existing method obtains is generally, and the inventive method to obtain fibrinogenic productive rate be 72%, show the increase of the fibrin of being gathered in the crops original 44%.
In preferred embodiments, fibrinogenicly be rich in hematoblastic blood plasma and contain at least 5 ten thousand platelet/mm from wherein being settled out 3, preferably contain the 200000/mm that has an appointment 3
The present invention can be in the relatively short time (about 20 minutes), from the anticoagulated whole blood of (20cc-150cc) relatively in a small amount, produce FVIII and protein concentrate (high), optimum fiber proteinogen, FXIII and platelet (result is that human growth factor increases) to more than 10 times.Raising in existing " Cell Saver " method of the present invention aspect the coagulation protein concentration makes it can produce the grumeleuse of more effective (tension force is bigger and shearing force is bigger) clinically.With respect to the used several blood that rise of " Cell Saver " method, the present invention can produce clinical effective dosage by the blood samples of patients of a small amount of (20cc-150cc) that simple venotomy known in the art obtained.
Method for optimizing adopts the special centrifugal machine and the detachable container that are described in the United States Patent (USP) 5707331 to handle the anticoagulated whole blood of gathering from patient's (or specified blood donors) on one's body.According to the present invention, the described method of above-mentioned patent is improved, be rich in hematoblastic blood plasma by suitably controlling centrifugal speed and the time length of centrifugal blood being provided.
To adopt venotomy and be distributed to first Room of the detachable container in 2-chamber, and an amount of precipitant such as PEG or saturated ammonium sulfate will be put into second Room from the anticoagulation that mammal is gathered on one's body.Above-mentioned ammonium sulfate can be the ammonium sulfate of 25-100%, and preferred about 95% ammonium sulfate.Above-mentioned detachable container is put into patent centrifuge described in United States Patent (USP) 5707331, and begin the described method of this patent.To this centrifugal programming to finish following steps automatically:
1. from whole blood, isolate erythrocyte under the speed of rotation that is rich in hematoblastic blood plasma (PRP) producing.This speed of rotation is called as " soft revolving ", and preferred that rotating speed that produces about 580G.Stop then centrifugal, and with PRP from the first Room impouring, second Room, in second Room with precipitant mix.Have been found that this " soft revolving " can produce and have about 50K/mm 3-450K/mm 3The blood plasma of PC.
2. after above-mentioned mixing is finished, restart centrifugally, precipitating proteins is concentrated described " revolving firmly " preferred that rotating speed that produces about 3500G by " revolving firmly " with platelet.
3. after above-mentioned the 2nd step, back first Room from second Room with the blood plasma Fibrinogen poorness and residual precipitant, stay relatively dry and be rich in the protein of somatomedin/platelet precipitation the platelet poorness.Have been found that and use the technology of using together than blood plasma (PPP) can gather in the crops more protein (particularly Fibrinogen) with PRP precipitant such as PEG or ammonium sulfate precipitant and platelet poorness.
4. proper amount of diluting (optimization citric acid salt buffer) is added, dissolve will precipitate once more, and results protein/platelet precipitation, so that for example carry with syringe.
5. when the condensing protein that contains the high-load human growth factor that will results mixes with thrombin and gained generation placed wound location, after placement, will form platelet gel in the several seconds.This gel is than using other conventional hemorrhage can obtain haemostatic effect faster.Because it has viscosity, it can also block air and fluid seepage, and because the existence of a large amount of platelet derived growth factors (PDGF) is arranged, the result causes healing faster.The performance of this gel comprises increasing of FVIII and Fibrinogen FXIII content, and human growth factor is than the improve of intrinsic level.The raising of these content of material makes the grumeleuse that is produced have better adhesion, tension force and shearing force.Because the content of desirable proteins is higher, has reduced the danger of grumeleuse failure before ripe, and increased the probability that obtains required result.
Embodiment 1
The 50ml whole blood is put into first Room of the container that is used for automatic centrifugal, and 15ml30% Polyethylene Glycol (MW1000) is put into second Room.Then with container about 3 minutes of about 580G " soft revolving ".Obtain 23-25ml thus and be rich in hematoblastic blood plasma, then this blood plasma is poured in second Room and made it and mix with PEG.Then, container is carried out centrifugal firmly, and supernatant backed in first Room.The result has obtained fibrinogen deposition, and its Fibrinogen productive rate is about 70%, and TGF-B-1 has increased 4-10 doubly, and PDGF-AB has increased by 30 times.
Embodiment 2
The 50ml whole blood is put into first Room of the container that is used for automatic centrifugal, and the 7ml saturated ammonium sulfate is put into second Room.Then with container about 3 minutes of about 580G " soft revolving ", and 23-25ml is rich in hematoblastic blood plasma pours in second Room, after being rich in hematoblastic blood plasma and ammonium sulfate mix, again container is carried out " revolving firmly " to obtain fibrinogen deposition, and supernatant backed in first Room, fibrinogenic productive rate in the gained precipitation is about 72%, and TGF-B-1 has increased 4-10 doubly, and PDGF-AB has increased by 30 times.
Modification within the scope of the appended claims is conspicuous for a person skilled in the art.

Claims (11)

1. separate the method for the fibrinogen concentrate that is rich in somatomedin, comprise the steps:
Hematoblastic blood plasma is rich in acquisition,
To described be rich in the hematoblastic blood plasma add the fibrinogen deposition agent and
Gather in the crops the fibrinogen concentrate that is rich in somatomedin the hematoblastic blood plasma from described being rich in.
2. according to the process of claim 1 wherein that described precipitant is a Polyethylene Glycol.
3. according to the process of claim 1 wherein that described precipitant is an ammonium sulfate.
4. according to the process of claim 1 wherein that step that hematoblastic blood plasma is rich in described acquisition comprises the step with centrifugal blood.
5. according to the method for claim 4, wherein said centrifugation step comprises makes described blood centrifugal about 3 minutes step under the centrifugal force of about 580G.
6. describedly be rich in hematoblastic blood plasma and comprise having 50,000-450,000 platelet/mm according to the process of claim 1 wherein 3Blood plasma.
7. make described hematoblastic blood plasma and the centrifugal together step of described precipitant of being rich in according to the process of claim 1 wherein that the fibrinogenic step of described results comprises.
8. according to the method for claim 1, the step that hematoblastic blood plasma is rich in wherein said acquisition comprises that the anticoagulated whole blood with about 50ml carries out the described step that is rich in hematoblastic blood plasma that centrifugal and decantation goes out 23-25ml, and the step of described adding precipitant comprises to described being rich in and adds the step that about 15ml 30% molecular weight is 1000 Polyethylene Glycol in the hematoblastic blood plasma.
9. according to the method for claim 1, the step that hematoblastic blood plasma is rich in wherein said acquisition comprises that the anticoagulated whole blood with about 50ml carries out the described step that is rich in hematoblastic blood plasma that centrifugal and decantation goes out 23-25ml, and the step of described adding precipitant comprises to the described step that adds about 7ml saturated ammonium sulfate in the hematoblastic blood plasma that is rich in.
10. according to the process of claim 1 wherein that the fibrinogenic step of described results also comprises the step that adds buffer in described Fibrinogen.
11. the product that makes by each described method among the claim 1-10.
CN98810252A 1997-10-17 1998-10-16 Precipitation of growth-factor-enriched fibrinogen concentrate from platelet rich plasma Expired - Fee Related CN1113656C (en)

Applications Claiming Priority (2)

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US6226497P 1997-10-17 1997-10-17
US60/062,264 1997-10-17

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CN1113656C true CN1113656C (en) 2003-07-09

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CA (1) CA2306629A1 (en)
WO (1) WO1999020288A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105708858A (en) * 2014-12-05 2016-06-29 国玺干细胞应用技术股份有限公司 Preparation method of growth-factor-platelet-rich fibrin and releasate

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EP1239894B1 (en) * 1999-12-22 2005-03-09 Henogen S.A. Bone generating product
CN1303413C (en) * 2003-06-17 2007-03-07 余伟明 Protein and virus quick-speed concentration method
KR20180110192A (en) * 2004-11-12 2018-10-08 바이엘 헬스케어 엘엘씨 Site-directed modification of fviii
CN1908005B (en) * 2006-08-11 2010-05-12 哈尔滨医科大学 Composite protein precipitator
EP2077118A1 (en) * 2008-01-07 2009-07-08 Gwo Rei Biomedical Technology Corp. Clottable concentrate of platelet growth factors and preparation method thereof
CN104711221B (en) * 2015-02-15 2017-09-15 第五空间健康管理江苏有限公司 Isolating immune cells and the method for extracting PRP are automated from adult peripheral blood
CN107198893A (en) * 2017-07-01 2017-09-26 张方亮 Leucocyte-removing PRP preparation method
CN107412878B (en) * 2017-08-07 2018-04-24 上海交通大学医学院附属第九人民医院 Composite fibrous scaffold and preparation method thereof
EP4206324A1 (en) * 2022-01-04 2023-07-05 Phynexus, Inc. Purification of macromolecules

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5030215A (en) * 1990-01-03 1991-07-09 Cryolife, Inc. Preparation of fibrinogen/factor XIII precipitate

Family Cites Families (4)

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Publication number Priority date Publication date Assignee Title
US5226877A (en) * 1989-06-23 1993-07-13 Epstein Gordon H Method and apparatus for preparing fibrinogen adhesive from whole blood
US5585007A (en) * 1994-12-07 1996-12-17 Plasmaseal Corporation Plasma concentrate and tissue sealant methods and apparatuses for making concentrated plasma and/or tissue sealant
US5510102A (en) * 1995-01-23 1996-04-23 The Regents Of The University Of California Plasma and polymer containing surgical hemostatic adhesives
US5707331A (en) * 1995-05-05 1998-01-13 John R. Wells Automatic multiple-decanting centrifuge

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5030215A (en) * 1990-01-03 1991-07-09 Cryolife, Inc. Preparation of fibrinogen/factor XIII precipitate

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105708858A (en) * 2014-12-05 2016-06-29 国玺干细胞应用技术股份有限公司 Preparation method of growth-factor-platelet-rich fibrin and releasate

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CN1276725A (en) 2000-12-13
EP1023078A1 (en) 2000-08-02
WO1999020288A1 (en) 1999-04-29
EP1023078A4 (en) 2004-09-29
CA2306629A1 (en) 1999-04-29
JP2001520198A (en) 2001-10-30

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