US3179567A - Reagent and method for determining the coagulability of blood - Google Patents

Reagent and method for determining the coagulability of blood Download PDF

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US3179567A
US3179567A US812682A US81268259A US3179567A US 3179567 A US3179567 A US 3179567A US 812682 A US812682 A US 812682A US 81268259 A US81268259 A US 81268259A US 3179567 A US3179567 A US 3179567A
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thromboplastin
plasma
cephalin
blood
coagulation
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Owren Paul Arnor
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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
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • 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
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2496/00—Reference solutions for assays of biological material
    • G01N2496/05—Reference solutions for assays of biological material containing blood cells or plasma

Definitions

  • Efiective anticoagulant treatment requires regular determinations of the coagnlability of the blood. It is necessary to adjust the dosage .to the individual case so as to avoid, on the one hand, excessive amounts which may result in bleeding complications, and,"on the other, too small a dosage which will fail to be effective.
  • Coagulation of the blood is effected by means of two partly separate coagulation systems: the external and the internal system.
  • the peroral anticoagulant agents cause a decrease in the concentration of the following four coagulation factors in the blood: prothrombin, Stuart-factor, pro-convertin and antihemophilia-B-factor.
  • prothrombin and Stuart-factor are involved in both systems; proconvertin acts only in the external system; and antihemophilia-B-factor participates only in the internal system. This is shown in the following scheme:
  • Coagulation system of the blood The "External System The Internal System (Cephalin Time) (Thromboplastin time) Blood platelet factor 3 (Cephalin), Antihemophilia-A-factor (Ali. G.) Antihemopl1ilia-Bfactor(P.T.C.). Antihemophilia-Ciaetor (P.T.A.). Hageman-iactor.
  • Prothrombin Proaceeleriu. Fibrinogeu.
  • FIGURE 1 shows. the sensitivity of these three methods to decreasing quantities of antihemophilia-B-factor. Concentration of antihemophilia-B- factor relative to the other factors isgiven as a percentage of the normal value along the abscissa. . The coagolation time in seconds (20 .to see.) is plotted on the ordinate.
  • Curve A shows the sensitivity of the new method to diminishing quantities of antihemophilia-B-factor.
  • the present invention provides a method which permits the practitioner to determine simultaneously the coagulability of the blood in both the external and the internal system with a single test.
  • the external and the internal coagulation system are started simultaneously and in such manner that .they proceed at approximately the same rate, with the result that the two systems exert fairly equal influences .onthe coagulation time of the system.
  • the method has undergone minor changes.
  • the latest detailed description is the following: Quick, A. 1.: Hemorrhagic Diseases, Lea and Fibiger, Philadelphia, 1957, pp. 379-387.
  • Thromboplastin suspension Extract of acetonedried rabbit brain. 7
  • 0.1 ml. oxalated plasma is mixed with 0.1 ml. thromboplastin solution, placed in water-oath at 37 C. and recalcified with 0.1 ml. of 0.01 M calcium chloride solution, and the coagulation time or the thromboplastin time (also called Quicks prothrombin time) is determined.
  • Thromboplastin and calcium are the only factors which are kept constant in Quicks method.
  • a change in any of the other factors of the external system: prothrombin, proconvertin, Stuart-factor, proaccelerin or fibrinogen, would also, however, be able to afieet the registered Quicks thromboplastin time.
  • proaccelerin and fibrinogen are relative- 1y constant; hence any prolongation of Quicks thromboplastin time will be due to a reduction of proconvertin, Stuart-factor, and/ or prothrombin.
  • the method is entirely insensitive to a reduction of antihemophilia-B-Factor. In hemophilia-B Quicks thromboplastin time?
  • Curve D shows the percentage of prothrombin found in the blood
  • curve E the percentage of proconvertin
  • curve F the percentage of antihemophilia-B-factor
  • the level of the three mentioned factors was approximately 100% at the beginning of the tests.
  • the anticoagulant agent phenylindandione was begun in daily doses, the quantity of which can be seen in the area at the top of the figure. On the left of this area the vertical line represents 100 mg. phenylindandione. The quantity of each days dose appears at the vertical line drawn from the date in question on the abscissa. On March the administration of the anticoagulant agent was stopped and the percentage content of the factors then rose quickly.
  • Thromboplastin suspension Aqueous extract of human brain.
  • the P-P-method is a determination of the thromboplastin time, i.e. it is primarily a method for estimating the activity of the external coagulation system.
  • the chief differences between the P-P-method and Quicks method are the following:
  • a new reagent, adsorbed bovine plasma is introduced in order to stabilize the concentration of proaccelerin and fibrinogen.
  • the measurement is therefore independent of the concentration of these factors in the test plasma. Since the factors which are reduced during anticoagulant treatment are relatively stable during storage of blood and plasma, the method may be used on stored samples. 7
  • a 10% dilution of the test plasma is used. This increases the sensitivity of the method, especially in the range 50-100%. Dilution of the test plasma also has the advantage that the risk of inoptimal calcium concentration with varying hematocrit values is far less than in Quicks method. The diluting also eliminates the effect of small amounts of heparin in the plasma to be examined.
  • the PAP-method is sensitive to reductions of prothrombin, Stuartfactor and proconvertin. [Gonyea, L.M., l-ljort, P., Owren, P.A.: The Journal of Laboratory & Clinical Medicine, 48, 624- 633 (1956).]
  • the thromboplastin used in the P-P-'nethod exerts, in addition to its thromboplastin effect, a cephalin effect on the internal coagulation system.
  • This effect is, however, not optimal.
  • the P-P-method possesses a certain, though exceedingly small, sensitivity to excessive reductions of antihemophilia-B-factor, if the external coagulation system is greatly impaired following anticoagulant treatment (see FIG. 1).
  • the following account describes the invention: a preparation which permits satisfactory determination of blood coagulability as a control of this treatment.
  • the method is based on a reagent which initiates both coagulation systems simultaneously and in such a way that they both require approximately the same amount of time.
  • the internal coagulation system has been given optimal conditions for maximum speed by inclusion of cephalin from human brains or any other cephalin preparation which has the same activity, in the reagent in optimal concentration.
  • the internal coagulation system alone will produce complete coagulation under these conditions in about 50 seconds.
  • the reagent contains a thromboplastin which starts the external coagulation system, but this thromboplastin is of such a nature that its maximal activity is low as compared with human brain thromboplastin and rabbit brain thromboplastin.
  • the speed of the external coagulation system is considerably reduced as compared to the normal speed obtained with active thromboplastin preparations of the type used for Quiclrs method and the P-P-method.
  • the thromboplastin used in this method should give a thromboplastin time with normal human plasma of 3550 seconds, as tested by Quicks method.
  • a weak thromboplastin could be produced by dilution of an active thromboplastin (of the type used for Quiclrs method and the P-Pmethod), but such a procedure results in a labile and practically useless reagent because small changes in its activity give considerable oscillations in the registered times.
  • the reagent therefore employs a thromboplastin which has a stable and low activity in optimal concentration and which has a relatively wide range of optimal concentrations. 7
  • thrombo- Jplastin As starting material for production of such a thrombo- Jplastin, lung or brain of certain species of animals (bovine, horse, swine, sheep, dog) are used.
  • the thromboplastin produced is species specific and reacts only slowly with human proconvertin, as revealed by the thromboplastin time of 35-50 seconds as compared with the thromboplastin time of 13-15 seconds of human brain thromboplastin.
  • PEG. 1 shows the marked sensitivity of this method to large reductions of antihemophilia-B-fac:tor, in contrast to the very defective sensitivity of Quicks method and the very slight sensitivity of the P-P-method.
  • the invention is, further, based on the concept that the coagulation times determined should be independent of changes in all the coagulation factors which are not affected by antocoagulant treatment. This is accomplished by the inclusion in the reagent of all these factors in high concentrations (actual concentrations specified below).
  • a specially adsorbed plasma (bovine, horse, swine, sheep, dog) prepared similar to that as for the P-l method, is used.
  • This adsorbed animal plasma contains high concentrations (minimal 90%relative to human plasma) of .antihernophilia- A-factor, antihemophilia-C-factor and Hageman-factor (of the internal system), and of proaccelerin and fabrinogen (which participate in both systems).
  • the reagent is specific for and sensitive to all the four factors which are reduced during anticoagulant treatment, and is at the same time uninfiuenced by variations in the other coagulation factors.
  • the coagulation times which are registered by this method therefore provide an adequate index of the total effect which the anticoagulant agents have on the coagulation of the blood.
  • Thedecisive difference from earlier methods is, as stated, the sensitivity of the new method to reduction of the concentration of antihemophilia- -factor.
  • composition of the reagent thus contains the following four components:
  • Thromboplostin Every type of :thromboplastin may he used, provided it fulfills the following requirements: (4) The thromboplastin suspension is stable on storage at to C. for at least 24 hours. (b) The optimal dilution of the thromboplastin suspension, which produces the maximal activity for the preparation as tested on normal human plasma, gives a thromboplastin time between 39 and seconds as tested by the method of Quick. This ,thromboplastin time provides the desired balance between the internal and the external clotting systems.
  • Thromboplastin preparations having the specified activity have beenprepared as follows:
  • Thromboplastin time Thromboplastin species of human plasma (seconds) Human brain thermoplastin, giving a thromboplastin time of 13-15 seconds, and rabbit brain thrombopiastin, producing a thromboplastin time of 12-13 seconds, cannot be used.
  • the bovine brain thromboplastin is most useful. A detailed technique of its preparation is given below. The optimal concentration of this thromboplastin giving maximal activity contains 1.3-1.7 g. of brain substance, weighed in the dried state, per 100 m1. of solution.
  • Cephalin All cephalin preparations may be used if they fulfill the following requirements: (a) The cephalin suspension must be stable for at least 24 hours at to 25 C. (b) The cephalin time produced with normal non-activated (prepared by silicon technique) platelet-poor [centrifuged for minutes at 3000 rpm. (1 ,800 g. at 4 C.)] human plasma must be shorter than seconds. [Technique as described from this laboratory by B. A. Waaler: Scand. J. of Clin. & Lab.
  • cephalin preparations have been tested and found suitable: (a) Crude cephalin prepared from human brain. (b) Cephalin prepared from soya beans.
  • the coagulation active principles in the cephalin preparations are phosphatidylethanolarnine, phosphatidylserine, and possibly other similar substances. There are great difiicult-ies in obtaining large quantities of individual phospholipid-s. The two first preparations are completely satisfactory for this reagent, however. Detailed techniques for (the preparation of crude human brain cephalin is given below.
  • the optimal concentration of crude cephalin in the final reagent is 0001-001 g. per 100 ml. of reagent and of asolectin 0010-0015 g. of dried substance per 100 ml. of reagent.
  • prothrornbin factor 11
  • proconverten factor V11
  • Stuart-ProWer-factor factor X
  • antihemophilic-Bfactor factor IX
  • Adsorbed plasma fulfilling the requirements given above have been prepared from bovine, horse, dog, sheep and human plasma.
  • Bovine plasma is preferred because of higher concentrations and greater stability of proaccelerin and antihcmophilia-A-tactor on storage.
  • the pH of the supernatant was adjusted to 7.35 with 0.5 normal NaOl-l, and volume of veronal bufier (pH 7.35 and ionic strength 0.154) was added.
  • This buffer was made by rnixing11.75 g. sodium diethyl barbiturate, 14.67 g. sodium chloride, 430 ml. 0.1 normal HCl and distilled Water to 2,000 ml.
  • the activity of a sample of the thromboplastin solution was tested in serial dilutions in physiological saline by Quicks method (0.20 ml. oxalated normal human plasma, 0.20 ml. thromboplastin solution, 0.20 ml.
  • the thromboplastin solution was consequently diluted with an equal volume of physiological saline solution.
  • the final solution contained 1.4 g. of dried brain substance per ml. of suspension.
  • the crude cephalin suspension is the acetone-insoluble, ether-soluble fraction of human brain. It was prepared as follows: Four hundred grams of human brain were Washed free of blood and membranes and ground with 300 ml. of acetone. (Merck & Company, Inc.) Afiter centrifugation, the acetone Was discarded and the residue re-extracted with the same volume of acetone a total of six times. The residue was then extracted with 1,800 ml. of ether overnight at room temperature. The ether was siphoned oil and evaporated to dryness by suction at 35 C. The resulting residue was washed twice for 15 minutes with 900 m1. of acetone and then redissolved in 200 m1. of ether.
  • the final reagent before freezedrying contained -7 of this thromboplastin suspension and consequently a concentration of 0.0056 g. percent cephalin.
  • the plasma is adsorbed with analytical grade BaSQ, (Baker) in an amount of 75 mg. per ml. of plasma for minutes and by slow stirring with a glass rod.
  • the plasma is dialyzed for 12 hours against ten times the plasma volume of 0.85 percent saline solution, the saline being changed three times (Cellulose Casings, Visiting Company, Chicago).
  • a 50 millimolar solution of calcium chloride is prepared by first mixing 50 ml. of the calcium chloride stock solution of 1 molar and 950 ml. of distilled water. One volume of this solution and 3 volumes of physiological saline solution are then mixed (this technique provides correct ionic strength).
  • Equal volumes of adsorbed bovine plasma and the thromboplastin cephalin suspension are mixed. To the mixture is added 6 its volume of a calcium chloride solution of 12.5 millimolar.
  • the freeze-drying of the mixture mentioned above is 10 performed in ampules of lenaglass of .30 ml. volume.
  • the ampules are of the usual type, capable of beingsealed *so as to preserve its contents.
  • the freeze-drying is then carried out under high vacuum and with supplying of heat to the ampule, for example so that the ampule is in contact with suitable elements, to 6070 C., so long as ice is still present in the mixture; and then the temperature is lowered to about 40 C. during the final drying step.
  • the product is treated under vacuum until the water content is below 0.5%.
  • the ampule is then sealed, preferably by closing the ampule opening by means of fusing undervacuum.
  • TheIfreeze-dried reagent is" reconstituted by addition :of distilled 'WEL'ECI or calciumchloride solution to the ampule for the capillary and venous blood method, respectively.
  • 0.5 ml. is placed by means of a pipette in a very small test tube (diameter 8-9 mm., length 60 mm.).
  • a measured quantity of the plasma or blood to be examined is added and the coagulation time determined with a stop-watch.
  • the recorded time is an expression of the effect of the anticoagulant treatment on the coagulation process.
  • the test is performed in a water-bath at 37 C.
  • the registered coagulation time is converted to relative activity, stated in percentage, using a correlation curve. Examples of correlation curves are given in FIG- URE 3. These correlation curves show the ratio between recorded coagulation times in two systems and the coagulation activity expressed in percentage of the normal.
  • the concentration of normal plasma in percentage is plotted on the logarithmic scale of the abscissa and the coagulation times in seconds (30-300 sec.) are plotted on the logarithmic scale of the ordinate.
  • Curve K shows the method used for capillary blood, 0.10 ml. volume.
  • Curve H shows the method used for citrate blood, 0.10 ml. volume, and for citrate plasma in dilution: 3 parts of plasma to 2 parts of saline,'and 0.10 ml. volume.
  • Curve C shows an alternative method used for citrate plasma in dilution: 1 volume of plasma to 4 volumes of saline, and 0.20 ml. of the dilution as test volume.
  • the method can be used both for capillary blood (whole blood), which contains no added anticoagulant, and for citrate blood or citrate plasma.
  • the reagent is provided with optimal concentration for capillary blood and cannot be used directly for citrate blood or citrate plasma, since for such samples the calcium concentration is inoptimal.
  • the dried reagent has to be reconstituted with 3.2 millimols calcium chloride solution which compensates for the amount of citrate in the test sample.
  • a skin incision which affords lively bleeding should be made in the finger tip or the ear lobe. 0.10 ml. of Whole blood is then filled into a pipette in the usual way, and this is at once mixed with the 0.5 ml. reagent which has previously been placed in the water-bath at 37 C. The coagulation time is determined.
  • Venous blood is used mixed with 3.13 percent (w./v.) sodium citrate dihydrate solution in the ratio 1 part sodium citrate solution to 9 parts of blood.
  • Example: 0.2 ml. sodium citrate solution is drawn into a 2 ml. syringe, and the syringe is then filled with blood by venipuncture to a volume of 2 ml. The sample is transferred to a small test tube and 0.10 ml. citrate blood is removed with a pipette and mixed with 0.5 ml. reagent, and the coagulation time is determined in the manner described above.
  • Citrate blood is prepared in the manner described in paragraph (2). After centrifuging, 0.6 ml. citrate plasma is 'measured off into a small test tube and 0.4 ml. physiological salt solution added. After mixing, 0.10 ml. of the diluted plasma is removed by pipette and at once mixed 'with .05 ml. of the reagent. The coagulation time is determined as indicated above.
  • the new all-in-one reagent constitutes a new creation in that it makes it possible to perform a combined deteri2 'mination of the coagulation activity of both the internal and the external coagulation systems.
  • the preparation of the reagent is based on principles which have not previously been employed.
  • the reagent has a number of advantages when compared with earlier reagents: (1) It is very stable at room temperature, thus simplifying distribution and storage. (2) It withstands heating up to 47 C. for a minimum of 30 days, and can therefore be used in tropical regions. (3) The requirement for only one eagent makes the procedure simpler and the performance of the test faster than was the case for earlier methods. (4) It is the only method so far devised which provides an adequate index of the'coagulation disturbance produced by oral anticoagulant agents. In contrast to earlier methods this one. is sensitive to reduction of antihemophilia-B-factor. (5) As a consequence of the above qualities, this method gives far greater protection against hemorrhagic complications than other methods. (6) The method is time-saving. (7) Because of the stability of the reagent in reconstituted form it affords reliable and reproducible results.
  • a combination of thrombopiastin from an animal organ and free cephalin in a single composition the thromboplastin being a member selected from the group consisting of brain throniboplastin and lug thromboplastin from at least one of bovine, swine, dog and sheep, the thromboplastin being stable on storage at from about to about C. for at least 24- hours and having a maximum activity on normal blood plasma to yield a thromboplastin time between and seconds, and the cephalin having a high reactivity toward the internal clotting system of human plasma to yield a cephalin time of less than seconds.
  • a composition for determining the coaguiability of blood for control of treatment with anticoagulant agents comprising as essential constituents the combination consisting essentially of ([1) animal organ thromboplastin which has a weak but constant reactivity toward the external clotting system of human blood plasma, the thromboplastin being a member selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, and (5) free cephalin having a high reactivity toward the internal clotting system of human plasma, the correlated proportions of the thromboplastin and the free cephalin assuring approximately cc al influence by both internal and external coagulation systems on coagulation time.
  • composition for determining the coagulability of blood for control of treatment with anticoagulant agents comprising:
  • .thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,
  • tuart-factor, proconvertin and antihemophilia- 13 B-factor have been removed, said plasma being a member selected from the group consisting of bovine, horse, swine, dog and sheep plasma, and
  • a blood coagulability-determining composition consisting essentially of (a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine; dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,
  • said plasma being a member selected from the group consisting of bovine, horse, swine, dog and sheep plasma, and
  • a blood coagulability-determining composition comprising:
  • thromboplastin selected from the group consisting of brain thromboplastinand lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma, and
  • the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.
  • a blood-coagulability-determining composition comprising:
  • thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma, and
  • the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.
  • a blood coagulability-determining composition comprismg:
  • thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,
  • a blood coagulability-determining composition comprising:
  • thromboplastin selected from the group consisting of brain thromboplastin and lung'thromboplastin from at least one of bovine, horse, swine, dog and sheep,the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,
  • the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal to the influence on coagulation time of both internal and external systems.
  • a blood coagulability-deternnning composition comprising:
  • thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,
  • the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.
  • a blood coagulability-determining composition comprising:
  • thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,
  • the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.
  • a blood coagulability-determining composition comprising:
  • thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,
  • adsorbed plasma from which plasma prothrombin, Stuart-factor, proconvertin and antihemophilia- B-factor have been removed, said plasma being a member selected from the group consisting of bovine, horse, swine, dog and sheep plasma,
  • a blood coagulabilityeterrnining composition comprisin (a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma, a (b) adsorbed plasma free from prothrornbin, Etuartfactor, proconvertin and antihernophilia-B-factor and containing a high concentration of all coagulation factors which are not affected by peroral anticoagulant agents, said plasma being selected from the group consisting of bovine, horse, swine, dog and sheep plasma, (c) free cephalin having a high reactivity toward the internal clotting system of human plasma, and (a') calcium chloride in an amount which gives optimal concentration for coagulation after reconstitution and addition of test plasma, the concentrations of said thromboplastin said free cephalin being correlated in the composition to maintain approximately equal the
  • a blood coagulability-determining composition comprising:
  • Tocantins The Coagulation of Blood, Grund and Stratton, New York, 1955, pp. 1789, 183484.

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3228841A (en) * 1965-06-10 1966-01-11 Warner Lambert Pharmaceutical Diagnostic reagent composition for determining blood coagulation factors and method of use
US3293134A (en) * 1961-09-08 1966-12-20 Warner Lambert Pharmaceutical Diagnostic reagent composition for determining blood coagulation factors and method of use
US3395210A (en) * 1964-02-07 1968-07-30 Warner Lambert Pharmaceutical Lyophilized diagnostic reagent for the determination of blood coagulation factors
US3486981A (en) * 1965-03-15 1969-12-30 Roy E Speck Substances relating to testing of blood-coagulation
US3880714A (en) * 1973-07-18 1975-04-29 Warner Lambert Co Diagnostic reagent
US3947378A (en) * 1974-12-23 1976-03-30 Warner-Lambert Company Adsorbed plasma
US4038147A (en) * 1976-06-15 1977-07-26 Becton, Dickinson And Company Method for detecting endotoxins
US4672030A (en) * 1984-02-28 1987-06-09 Peter Witt Test kit for the determination of activated thromboplastin time (PTT) with increased sensitivity to heparin
EP0241613A1 (fr) * 1986-04-17 1987-10-21 Bio/Data Corporation Réactif microconcentré en forme de tablettes pour la coagulation de thromboplastine dans le plasma du sang et procédé pour sa préparation
US4851336A (en) * 1985-09-05 1989-07-25 Yin E Thye Composition, kit, and method for assaying heparinano a method for making the composition
US4946775A (en) * 1985-09-05 1990-08-07 Yin E Thye Composition, kit and method for assaying heparin and a method for making the composition
US4948724A (en) * 1985-09-05 1990-08-14 Yin E Thye Composition, kit and method for assaying heparin and a method for making the composition
WO1992008479A1 (fr) * 1990-11-13 1992-05-29 Corvas International, Inc. Reactif de temps de prothrombine base sur la thromboplastine tissulaire
US6203816B1 (en) * 1990-11-13 2001-03-20 Corvas International, Inc. Tissue factor based prothrombin time reagent
AU744287B2 (en) * 1990-11-13 2002-02-21 Corvas International, Inc. Tissue factor based prothrombin time reagent
US6596543B2 (en) 2001-03-22 2003-07-22 Dade Behring Inc. Use of liposomes of defined composition and size for the preparation of prothrombin time reagents
US6706861B2 (en) 1999-12-10 2004-03-16 Dade Behring, Inc. Reconstitution of purified membrane proteins into preformed liposomes
US20120231485A1 (en) * 2011-03-08 2012-09-13 Oenundarson Pall Torfi Method for monitoring anticoagulant therapy

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4002739A (en) * 1975-02-18 1977-01-11 Warner-Lambert Company Soluble collagen
DE2915310A1 (de) * 1979-04-14 1980-10-30 Behringwerke Ag Neues partielles thromboplastin und dessen verwendung
DE3150596A1 (de) * 1981-12-21 1983-06-30 Boehringer Mannheim Gmbh, 6800 Mannheim Verfahren zur herstellung von gewebsthromboplastin
DE3413311A1 (de) * 1984-04-09 1985-10-17 Behringwerke Ag, 3550 Marburg Reagenz zur bestimmung der thromboplastinzeit
JP7110360B2 (ja) 2017-10-09 2022-08-01 テルモ ビーシーティー バイオテクノロジーズ，エルエルシー 凍結乾燥方法
JP7471316B2 (ja) 2019-03-14 2024-04-19 テルモ ビーシーティー バイオテクノロジーズ，エルエルシー マルチパート凍結乾燥容器
CN112481355B (zh) * 2020-11-16 2023-05-30 武汉市长立生物技术有限责任公司 液体型凝血酶原时间测定试剂盒及其制备方法

Citations (2)

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Publication number Priority date Publication date Assignee Title
US2687980A (en) * 1948-09-15 1954-08-31 Ernest W Blanchard Thromboplastin product and method for preparing same
US2847350A (en) * 1954-05-27 1958-08-12 Ortho Pharma Corp Preparation of highly active thromboplastin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2687980A (en) * 1948-09-15 1954-08-31 Ernest W Blanchard Thromboplastin product and method for preparing same
US2847350A (en) * 1954-05-27 1958-08-12 Ortho Pharma Corp Preparation of highly active thromboplastin

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3293134A (en) * 1961-09-08 1966-12-20 Warner Lambert Pharmaceutical Diagnostic reagent composition for determining blood coagulation factors and method of use
US3395210A (en) * 1964-02-07 1968-07-30 Warner Lambert Pharmaceutical Lyophilized diagnostic reagent for the determination of blood coagulation factors
US3486981A (en) * 1965-03-15 1969-12-30 Roy E Speck Substances relating to testing of blood-coagulation
US3228841A (en) * 1965-06-10 1966-01-11 Warner Lambert Pharmaceutical Diagnostic reagent composition for determining blood coagulation factors and method of use
US3880714A (en) * 1973-07-18 1975-04-29 Warner Lambert Co Diagnostic reagent
US3947378A (en) * 1974-12-23 1976-03-30 Warner-Lambert Company Adsorbed plasma
US4038147A (en) * 1976-06-15 1977-07-26 Becton, Dickinson And Company Method for detecting endotoxins
US4672030A (en) * 1984-02-28 1987-06-09 Peter Witt Test kit for the determination of activated thromboplastin time (PTT) with increased sensitivity to heparin
US4851336A (en) * 1985-09-05 1989-07-25 Yin E Thye Composition, kit, and method for assaying heparinano a method for making the composition
US4948724A (en) * 1985-09-05 1990-08-14 Yin E Thye Composition, kit and method for assaying heparin and a method for making the composition
US4946775A (en) * 1985-09-05 1990-08-07 Yin E Thye Composition, kit and method for assaying heparin and a method for making the composition
US4755461A (en) * 1986-04-17 1988-07-05 Bio/Data Corporation Tableted blood plasma microconcentrated thromboplastin coagulation reagent
JPS62250364A (ja) * 1986-04-17 1987-10-31 バイオ／デ−タ・コ−ポレイシヨン 改良された錠剤化血漿マイクロ濃縮トロンボプラスチン凝固試薬及びその製法
EP0241613A1 (fr) * 1986-04-17 1987-10-21 Bio/Data Corporation Réactif microconcentré en forme de tablettes pour la coagulation de thromboplastine dans le plasma du sang et procédé pour sa préparation
WO1992008479A1 (fr) * 1990-11-13 1992-05-29 Corvas International, Inc. Reactif de temps de prothrombine base sur la thromboplastine tissulaire
US5314695A (en) * 1990-11-13 1994-05-24 Corvas International, Inc. Tissue factor based prothrombin time reagent
US6203816B1 (en) * 1990-11-13 2001-03-20 Corvas International, Inc. Tissue factor based prothrombin time reagent
AU744287B2 (en) * 1990-11-13 2002-02-21 Corvas International, Inc. Tissue factor based prothrombin time reagent
US6706861B2 (en) 1999-12-10 2004-03-16 Dade Behring, Inc. Reconstitution of purified membrane proteins into preformed liposomes
US6596543B2 (en) 2001-03-22 2003-07-22 Dade Behring Inc. Use of liposomes of defined composition and size for the preparation of prothrombin time reagents
US20120231485A1 (en) * 2011-03-08 2012-09-13 Oenundarson Pall Torfi Method for monitoring anticoagulant therapy
US9234902B2 (en) * 2011-03-08 2016-01-12 Pall Torfi Önundarson Method for monitoring anticoagulant therapy

Also Published As

Publication number Publication date
ES249137A1 (es) 1959-11-16
NL123584C (fr)
DE1189763B (de) 1965-03-25
NL238873A (fr)
FR1223856A (fr) 1960-06-21
CH408283A (de) 1966-02-28
GB917012A (en) 1963-01-30

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