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WO1999060409A1 - Fibrinopeptides salivaires utilises comme indicateurs de la coagulabilite sanguine - Google Patents

Fibrinopeptides salivaires utilises comme indicateurs de la coagulabilite sanguine Download PDF

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Publication number
WO1999060409A1
WO1999060409A1 PCT/IB1999/000869 IB9900869W WO9960409A1 WO 1999060409 A1 WO1999060409 A1 WO 1999060409A1 IB 9900869 W IB9900869 W IB 9900869W WO 9960409 A1 WO9960409 A1 WO 9960409A1
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Prior art keywords
saliva
blood
combined
fpa
coagulation
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PCT/IB1999/000869
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English (en)
Inventor
Matteo De Nora
Jacob R. Blasius
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Link Enterprises Corp
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Link Enterprises Corp
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Priority to US09/673,085 priority Critical patent/US6503724B1/en
Priority to AU36226/99A priority patent/AU3622699A/en
Publication of WO1999060409A1 publication Critical patent/WO1999060409A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin

Definitions

  • Blood coagulation is a complex system involving a large number of proteins that function in concert to yield hemostasis.
  • the coagulation system is regulated by a series of proteins present in plasma and on the surface of cells.
  • pro- and anti-coagulant mechanisms are delicately balanced to provide hemostasis and coagulation. Disturbances in this balance result in either bleeding or thromboembolic disorders, and can be induced by medical conditions, congenital or acquired, the intake of drugs or vitamins.
  • the most widely used tests to measure the coagulation status of a person are known as clotting assays. Clotting assays measure the clotting times of recalcified platelet-poor plasma.
  • the prothrombin time (PT) test is performed by adding calcium and thromboplastin to citrated plasma.
  • thromboplastin refers to a phospholipid- protein extract of tissues, usually lung, brain, or placenta, that contains both tissue factor and the phospholipid necessary to promote the activation of factor X by Factor VII.
  • the standard thromboplastin is "The World Health Organization" (WHO) thromboplastin which is derived from human brain thromboplastin.
  • WHO World Health Organization
  • PT test instruments are based upon measuring how the plasma viscosity changes over time after calcium and thromboplastin have been added to plasma. When plasma coagulates it turns from a solution to a gel which is more viscous.
  • decalcified plasma is incubated for a specified time with activating agents such as ellagic acid, silica, soy extract, or kaolin. Following incubation with the activating substance, calcium chloride is added to the plasma mixture for a clot to form.
  • activating agents such as ellagic acid, silica, soy extract, or kaolin.
  • plasma is incubated for 3 minutes with a reagent supplying procoagulant phospholipid and surface-active powder (e.g. micronized silica). Calcium is then added and the clotting time noted.
  • a reagent supplying procoagulant phospholipid and surface-active powder e.g. micronized silica.
  • clotting assays present several drawbacks.
  • clotting assays are by definition invasive, as they require some blood to be drawn from the patient.
  • Third, clotting assays depend on an additional product, thromboplastin, whose performance can be different from the reference WHO thromboplastin, requiring complicated International Normalization Ratio (INR) calculations to ensure accuracy.
  • ILR International Normalization Ratio
  • clotting tests are indirect tests, reflecting only indirectly fibrinopeptides and fibrinogen degradation products and other proteins related to the state of anticoagulation.
  • the purpose of this invention is to use a non invasive diagnostic test technique to measure the level of blood anticoagulation induced by pharmaceuticals which prevent or reduce coagulation of blood as well as to measure and/or identify any natural or disease-induced blood disorders which effect the coagulation of blood.
  • the invention can be used to measure the level of hypercoagulation induced by drugs, proteins and vitamins which augment coagulation of blood (procoagulant drugs).
  • the invention is a non invasive test since it measures key substances present in saliva, filtered saliva, sputum, or the like, which are obtainable non invasively (hereunder referred to as "saliva").
  • the invention is usable in humans as well as in animals.
  • saliva and urine are very different in terms or biological roles and compositions, as well as in their enzymatic environments.
  • Examples of clinical applications in which the test could be used include all blood coagulation disorders, be they congenital, acquired, or drug-induced.
  • congenital deficiencies of the intrinsic pathway of the coagulation system (hemophilia A and B, deficiencies of Fitzgerald factor and Fletcher factor), deficiencies in protein C and protein S, heparin and heparin-like therapy, Warfarin (Coumadin)-like therapy, acute thrombotic situations such as acute myocardial infarction or pulmonary embolism, Vitamin K deficiency or excess, hypofibrinogenemia, liver disease, disseminated intravascular coagulopathy, among others.
  • the invention produces a quantitative measurement of prothrombin fragment 1 +2 (F1 +2), prothrombin fragment 1 (F1), prothrombin fragment 2 (F2), fibrinopeptide A (FpA) and D-Dimers in saliva which is correlated with the coagulation time as expressed for example by prothrombin time (PT), INR, partial thromboplastin time (PTT) and activated partial thromboplastin time (APTT).
  • Fragments F1 +2, F1 , F2, FpA and D-Dimers are preferably measured by means of immunoassay techniques, which measure a substance using the reaction of an antibody (immunoreagent) with an antigen (i.e. the protein to be measured).
  • the quantification of the substance measured is determined by measuring how much of the antigen is bound to the antibody and how much of the antigen is not bound to the antibody.
  • Enzyme-labeled, fluorescent-labeled, phosphorescent-labeled, radiolabeled, chemiluminescent-labeled and bioluminescent-labeled immunoassay techniques are for example usable to measure the concentrations of Fragments F1+2, F1 , F2, FpA and D-Dimers.
  • Capillary action, precipitation, turbidometric, diffusion, agglutination and electrophoretic immunoassay techniques can also be used in practicing the invention, as well as potentiometric, amperomethc, piezoelectric and evanescent-wave immunosensors.
  • any combination of the aforesaid assay techniques can also be employed to measure the concentrations of Fragments F1+2, F1 , F2, FpA and D-Dimers in saliva.
  • a preferred application of this invention is to control the level of any pharmaceutical which results in anticoagulation of the blood by affecting the conversion of blood Factor X to Factor Xa, which is commonly termed activated Factor X.
  • Factor X and Factor Xa which are in the latter stages of the coagulation cascade best correlate with the anticoagulation effect (see FIGURE 1 for diagram of coagulation cascade).
  • Factor X is a glycoprotein of molecular weight 55,000 and composed of two polypeptide chains linked by one disulfide bond. The light and heavy chains have molecular weights of 16,000 and 38,000 daltons respectively.
  • Factor X is converted to Factor Xa by either factors IXa, and VIII or Factor VII and tissue Factor.
  • Factor X can also be activated by other proteases such as trypsin.
  • the activation of Factor X by each of these systems involves the cleavage of a single specific arginyl-isoleucine peptide bond in the heavy chain of Factor X. This gives rise to the formation of a glycoprotein of molecular weight 44,000 and a peptide with a molecular weight of 11 ,000.
  • a common feature of Fragments F1+2, F1 , F2, FpA and D-Dimers as measured in the invention is that their concentration rises when the conversion of Factor X to Factor Xa increases, and decreases when the conversion of Factor X to
  • Activated blood factor X forms a complex with activated blood factor V, phospholipid and calcium.
  • the first step in the activation by the complex is the proteolytic cleavage of a fragment from the NH2-terminal end of prothrombin. This gives rise to what is termed intermediate II (an intermediate precursor of thrombin) and the clipped fragment referred to as prothrombin fragment 1 +2.
  • Intermediate II (which is a single chain polypeptide) is then cleaved a second time by activated factor X complex to yield a two chain (light and heavy chains) thrombin molecule.
  • Thrombin can use prothrombin as a substrate.
  • the cleavage pattern differs from that obtained by the activated factor X complex.
  • Thrombin cleaves prothrombin releasing a fragment smaller than that cleaved by the activated factor X complex. This gives rise to what is termed prothrombin fragment 1 and intermediate I. This cleavage occurs without the involvement of activated factor V, phospholipid or calcium.
  • Thrombin cannot activate prothrombin to thrombin.
  • Activated factor X is required for the conversion to thrombin; however, activated factor X then cleaves off the remaining portion of prothrombin fragment 1 +2 from intermediate I to give rise to fragment 2 and intermediate II.
  • FIGURE 3 shows the generation of FpA by Thrombin.
  • Fibrin clot Formation is initiated when thrombin cleaves fibrinopeptides A and B from the E domain of fibrinogen.
  • the resulting soluble fibrin monomers align in a staggered end-to-end arrangement to produce protofibhls. Protofibrils later polymerize side-by-side into large, soluble fibrin oligomers.
  • fibrinogen degradation products FDP. Plasmin cannot distinguish between fibrinogen and fibrin : therefore, it degrades both. This results in the appearance of essentially the same fragments from fibrinogen and fibrin.
  • FIGURE 4 shows the sequence of the reactions in the degradation of fibrinogen and fibrin by plasmin and the principal products, fragment X,Y,D (D-D dimer), and E.
  • Fragments X and Y are referred to as the early degradation products.
  • Fragments D and E are the late degradation products.
  • Fragment X is the first and largest fragment formed (250,000 Daltons).
  • Fragment X is the result of plasmin cleavage of the terminal portion of the alpha chains from a fibrin polymer, leaving isolated fibrin stands.
  • Fragment X is then cleaved by plasmin to form two fragments called Y (YY) and an intermediate complex DXD. This complex is further cleaved into intermediate complexes DED and DY/YD until finally fragments E and D (D-D dimer) are formed.
  • any pharmaceutical which reduces the conversion of Factor X to Factor Xa will reduce the level of Fragment 1 +2, F1 , F2, FpA and D-Dimer in body fluids. Reductions in Factor Xa result in blood taking a longer time to coagulate. Therefore, the quantity of Fragment 1+2, F1 , F2, FpA and D-Dimer is inversely proportional to the level of anticoagulation. The less Fragment 1+2, F1 , F2, FpA and D-Dimer in saliva, the longer it will take for blood to coagulate. Conversely, the more Fragment 1+2, F1 , F2, FpA and D-Dimer in saliva, the less time it will take the blood to coagulate.
  • - gingival crevicular fluid may contain blood, - saliva flow rate varies due to many factors (for example stress and degree of hydration).
  • the transport of coagulation markers to saliva is a more direct route from blood than urine.
  • the urine is stored in the bladder before excretion and this is a major drawback of urine use as a diagnotic medium.
  • the invention is relating as well to a non invasive device for doing in-vitro analysis of the concentration of the coagulation markers which best correlate to the state of anticoagulation of individuals undergoing Warfarin therapy so that the individuals can control their Warfarin dosage, thereby reducing visits to care-giver offices and increasing the accuracy of dosing.
  • the functional requirements of the test are : be non invasive; not require a blood sample to be withdrawn from a user; be able to be used at home by persons undergoing Warfarin therapy; be able to be carried out by persons of average intelligence and motor skills after receiving minimal training; results to be readable by the users eye or an inexpensive device such as a photometer, densitometer or colorimeter; be cost effective in that the cost of the test is to be less than the cost charged by physicians and clinics for the patient visit and the cost of the test procedure; be capable of accurately measuring for a person which is stabilized on Warfarin therapy, the coagulation markers over the expected range in the saliva being analyzed; not require meticulous attention to specimen acquisition and processing; only require "spot" saliva; portable due to small size and weight; provides rapid results, preferably less than 15 minutes; provides a quantitative result, preferably in the form of a permanent record.
  • An at home test saves time and money, helps those patients which have a hardship traveling to institutions, the debilitated and those lacking adequate transportation.
  • An at home test also improves anticoagulation therapy and safety as self monitoring has proven more accurate than institution based monitoring, especially for patients interested in their well being. It induces patients to have a greater responsibility for managing one's own therapy, allows for more frequent monitoring, especially when fluctuating PT values require adjusting of the anticoagulant dose.
  • Figure 1 is a diagram showing the coagulation cascade.
  • Figure 2 is a diagram showing the Prothrombin to Thrombin conversion pathway.
  • Figure 3 is a diagram showing the generation of FpA by Thrombin.
  • Figure 4 is a diagram showing the generation of D-Dimers during degradation of Fibrin polymers by plasmin.
  • Figure 5 is a diagram showing the principle of an enzymoimmunoassay as used in practicing the invention.
  • fragments F1+2, F1 , F2, FpA, and D-dimers are measured by immunoassay technology, preferably enzymoimmunoassay technology, in which a labeled antibody is used to determine the amount of protein (antigen) in body fluids.
  • a known amount of antigen here fragment F1 +2, F1 , F2, FpA, D-dimers
  • the body fluid to be tested containing the antigen to be measured (F1+2, F1 , F2, FpA, D-dimers) is then put in contact with the substrate.
  • This method depends on the distribution of a limited amount of enzyme-labeled antibody, which is added to the body fluid, between the antigen on the substrate and the antigen in body fluid. Maximum binding of the labeled antibody to the immobilized antigen will take place in the absence of antigen to be measured. As the concentration of antigen to be measured in the body fluid increases, the amount of labeled antibody which will attach to the antigen bound to the substrate will proportionally decrease. This result is due to a portion of the labeled antibody attaching to the immobilized antigen as well as the antigen in body fluid (see Figure 5). Measurement of the amount of antibody attached to the antigen which is bound to the substrate allows for the calculation of the amount of antigen which is in the body fluid.
  • FpA FpA One of the commercially available FpA antibodies produced by Boeh nger Mannheim or any other manufacturer of FpA antibody
  • D-Dimer D-Dimer DD-36B monoclonal antibody produced by Dade, Miami, Florida or any other manufactures of D- Dimer antibody This ELISA employees an antigen attached to the substrate.
  • the following examples will show, that an ELISA in which an antibody is attached to the substrate may be employed as well.
  • a preferred embodiment of the invention is to measure F1 +2, F1 , F2, FpA,
  • a group of 13 individuals undergoing Warfarin anticoagulation therapy (®Coumadin which is manufactured by DuPont Merck Pharmaceuticals) for the medical conditions of atrial fibrillation and mechanical heart values were tested for the concentration of the coagulation markers in their plasma and saliva.
  • Warfarin is given just as an example for an anticoagulant therapy; the scope of the invention extends to all similar anticoagulant therapies and of course any procoagulant therapy.
  • Class # 2 Subjects with atrial fibrillation receiving long term Warfarin therapy and being controlled to an anticoagulation level of 2.0-3.0 INR. These subjects were patients which were normally having their prothrombin time checked every 1 to 3 weeks.
  • Class # 3 Subjects with atrial fibrillation receiving Warfarin therapy and being controlled to an anticoagulation level of 2.0-3.0 INR. These subjects were patients which have been stabilized on Warfarin therapy for one month or more, but were expected to end their therapy in 3 to 4 months. These subjects were patients which were normally having their prothrombin time checked every 1 to 3 weeks. Alternately, these subjects could be individuals which were beginning Warfarin therapy. If they were beginning Warfarin therapy, they were included in the study if they had not been undergoing anticoagulation therapy or thrombolytic therapy in the previous 5 days.
  • Class # 1 and # 2 The purpose of Class # 1 and # 2 was to measure the concentration of coagulation markers for subjects which are stabilized on long term Warfarin therapy.
  • Class # 3 was to measure how the concentration of the coagulation markers change as the subjects which were stabilized are then taken off Warfarin therapy or conversely how the concentration of the coagulation markers change as the subjects are initiated and then stabilized on Warfarin therapy.
  • the subjects followed the a specific diet and living habit regiment during the study period, with no medications and illicit drug use, with particular attention to drugs potentiating Warfarin's anticoagulation effect and drugs inhibiting Warfarin's anticoagulation effect. They were tested for illicit drug use using the SmithKline Drug Abuse Panel 10-50.
  • the subjects provided blood, urine and saliva specimens sequentially on the same day usually in the morning, always within plus or minus 1 hour of a regularly scheduled time (e.g. if on the subject's first day of providing a specimen the specimen collection was at 11 :00 AM then every succeeding time a specimen was provided it was between 10:00 AM and 12:00 noon).
  • This time constraint was imposed to minimize the effects of the circadian rhythm of a person's coagulation system. Of the 89 sets of specimens collected only 7 were not collected according to the time constraint.
  • Two 4.5 ml venous blood specimens were collected each time the subjects provided blood specimens.
  • One blood specimen was used for the determination of prothrombin time within 24 hrs.
  • the other blood specimen was for determination of coagulation marker concentrations.
  • Normal venous blood collection procedures were used. Each blood specimen was collected during the same visit and within moments before or after the saliva or urine specimens were collected. Each blood specimen was collected with a Becton Dickinson Coagulation Vacutainer tube with siliconized interior (catalog # 366415) containing 0.5 ml 0.105 M buffered sodium citrate anticoagulant.
  • the blood was collected per NCCLS H3-A2 procedures so as to have 9Vol venous blood and 1Vol sodium citrate. Care was taken to not force blood from the subjects' veins. Within 1 hour of sample collection the whole blood was centrifuged for 10 minutes at 3000 rpm. The supernatant plasma was withdrawn and immediately stored at -20 degrees centigrade.
  • the subjects were first asked if his gums have been bleeding or if his gums bled the last time subject brushed his teeth so as to avoid collecting saliva which may have contained blood or saliva which may have excessive amounts of fluid from the gingival crevice.
  • No saliva samples were collected from subjects with bleeding gums. Two saliva samples were simultaneously collected. Approximately 2 ml of saliva was collected from each subject.
  • the saliva collection device is the Saliva Sampler manufactured by Saliva Diagnostic Systems. The device consists of a collection tube, a cotton collector pad with a built in indicator and a filter (Porex Blood Serum Filter) which separates out cellular matter from the whole saliva.
  • the saliva collection tube as supplied by the manufacturer contains a preservative transport buffer. This solution was removed and the collection tubes were washed and sterilized before being used.
  • the collector pad collects between 0.8 to 1.3 ml of whole saliva in 3 minutes (normal range 1-7 minutes).
  • the collector pad assembly has a built in indicator which turns color from white to blue when the collector pad is saturated.
  • the saliva which was collected is the saliva from the sublingual gland and submaxillary gland located under the tongue at the floor of the mouth. Contact with the gingival crevice (area between the gums and the tooth) was avoided. The collection technique was unstimulated saliva collection. That is the saliva being collected was with the subject not chewing or otherwise stimulating the inside of the mouth.
  • Porex filter was inserted into the collection tube and the filtered saliva was then poured into a cryotube. The cryotube was immediately frozen at -20°C.
  • the amount of filtered saliva recovered by the above procedure was between 0.250 ml and 0.50 ml per collection pad.
  • the blood samples collected from the clinical trial subjects were immediately frozen at -20°C. Within 24 hours of blood sample collection, the samples prothrombin time and INR were measured in the laboratory. All INR results were obtained by using the same lot of thromboplastin on the same PT test equipment.
  • the test equipment and thromboplastin are manufactured by Organon Teknika.
  • the control group PT reference range is 12.1 to 14.5 seconds with 12.8 seconds used for all PT calculations.
  • the important points are that the patients were stabilized on Warfarin and they were free of diseases and drugs which would interfere with the therapy and cause blood to be in the urine or saliva.
  • the testing of the saliva to determine the concentration of the coagulation markers was conducted with ELISA.
  • the ELISA test only requires 0.100 miliiliters of filtered saliva. It is best to collect 0.250 miliiliters of filtered saliva so that the test can be run in duplicate which is standard procedure with ELISA tests.
  • F1 antibody to human prothrombin fragment 1 , F1 , antigen was developed by injecting human prothrombin fragment 1 into a New Zealand White rabbit. The rabbit then produces an F1 antibody which is a polyclonal antibody.
  • New Zealand White rabbits were injected intradermally with 1 ml human prothrombin fragment 1 antigen emulsified with Freund's complete adjuvant on day 1. On days 6 and 13 the rabbits were intradermally injected with Freund's incomplete adjuvant. Intra-muscular booster injections were repeated once per month thereafter. The rabbit serum was harvested 6 weeks after the initial injection and stored at -20°C.
  • the first step is to grossly purify the rabbit serum containing the F1 antibody by the protein A purification method, (Bollag, D.M., et. al., 1966, Protein Methods, 2nd Ed. 415 pages, Wiley-Liss, NY, NY).
  • An affinity column containing Affi-Gel Protein A Agarose is prepared.
  • the rabbit serum is processed through the column which binds the F1 antibody and the other antibodies in the serum.
  • the F1 antibody and other antibodies are recovered from the column with an elution buffer.
  • the second step is to dialyze the solution obtained in step 1 against 3 X 2 liters of PBS.
  • the dialyzed solution is then passed through a F1 affinity column (the affinity column contains F1 antigen) 10 times to capture the F1 antibody contained in the solution.
  • the F1 affinity column is washed with PBS to remove the unbound and nonspecifically bound antibodies.
  • Actisep elution medium (Sterogene Bioseparations, cat. # 9701-01) is added to the column and incubated for 10 minutes at room temperature. The eluate which contains the F1 antibody is collected for additional processing.
  • the third step is to remove the antibodies which will cross react with F2 antigen.
  • the solution obtained in step 2 is passed through an affinity column containing F2 antigen. Ten passes are performed to remove the antibodies which cross react with F2.
  • the final solution is a purified F1 polyclonal antibody which has no significant cross reactivity with F2 antigen.
  • the ELISA uses the purified F1 antibody bound to a solid substrate as the capture antibody to remove F1 antigen from saliva specimens.
  • the detection antibody is F1 antibody labeled with Horseradish Peroxidase, HRP.
  • F1 antibody is labeled with HRP to produce the detection antibody which attaches to the F1 antigen which is captured from the saliva specimen by the F1 antibody coated to the ELISA plate.
  • HRP is covalently attached to the F1 antibody using amino and thiol directed cross-linkers per procedures outlined in Ishikawa, E., et.al., 1983, "Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining", J. Immunoassay 4: 209-327.
  • This procedure describes how to test a saliva specimen for the concentration of F1 antigen contained in the saliva :
  • the following data show the correlation between prothrombin time as expressed by INR or various coagulation conditions and plasma concentrations of the fragments, as well as the correlation between concentrations of the fragments in plasma, saliva, and urine.
  • the concentration of the fragments in saliva being directly correlated to the concentration of the same in plasma, one can infer the coagulation status of an animal or human by using the method of the invention on a saliva sample.

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Abstract

L'invention concerne une technique non invasive destinée à déterminer la coagulabilité du sang chez l'homme ou l'animal. On dose dans la salive, dans les expectorations, etc., les facteurs F1+2, F1, F2, FpA, les dimères D, F1+2 combiné à F1, ou F1+2 combiné à F2, dont les concentrations respectives sont inversement proportionnelles au temps que met le sang à coaguler et au risque d'hémorragie, et directement proportionnelles au risque de formation in vivo de thrombus ou d'emboles. Cette technique peut être utilisée dans n'importe quel trouble de la coagulation, qu'il soit congénital, acquis on iatrogène.
PCT/IB1999/000869 1998-05-18 1999-05-14 Fibrinopeptides salivaires utilises comme indicateurs de la coagulabilite sanguine Ceased WO1999060409A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US09/673,085 US6503724B1 (en) 1998-05-18 1999-05-14 Fibrinopeptides in saliva as indicator of blood coagulation status
AU36226/99A AU3622699A (en) 1998-05-18 1999-05-14 Fibrinopeptides in saliva as indicator of blood coagulation status

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EP98810460 1998-05-18
EP98810460.0 1998-05-18
IB9800777 1998-05-19
IBPCT/IB98/00777 1998-05-19

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001063299A1 (fr) * 2000-02-23 2001-08-30 Besst-Test Aps Methode de correlation de l"activite de coagulation sanguine avec des marqueurs dans des liquides biologiques, par exemple l"urine
US6503724B1 (en) * 1998-05-18 2003-01-07 Link Enterprises Corporation Fibrinopeptides in saliva as indicator of blood coagulation status
US7473548B2 (en) 2003-04-25 2009-01-06 Medtronic, Inc. Optical detector for enzyme activation
WO2011049431A1 (fr) * 2009-10-19 2011-04-28 Stichting Katholieke Universiteit, Radboud Universiteit Nijmegen Mesure du statut de coagulation sanguine dans la salive
EP3422011A1 (fr) * 2017-06-28 2019-01-02 Koninklijke Philips N.V. L'estimation de valeur de paramètre dans un système de coagulation

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RU2110070C1 (ru) * 1994-10-06 1998-04-27 Морозова Татьяна Ивановна Способ дифференциальной диагностики туберкулеза легких и пневмонии

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Cited By (10)

* Cited by examiner, † Cited by third party
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US6503724B1 (en) * 1998-05-18 2003-01-07 Link Enterprises Corporation Fibrinopeptides in saliva as indicator of blood coagulation status
WO2001063299A1 (fr) * 2000-02-23 2001-08-30 Besst-Test Aps Methode de correlation de l"activite de coagulation sanguine avec des marqueurs dans des liquides biologiques, par exemple l"urine
JP2003524187A (ja) * 2000-02-23 2003-08-12 ベッスト−テスト アンパーツゼルスカブ 体液、例えば尿におけるマーカーと血液凝固活性とを相互関連づけるための方法
US7473548B2 (en) 2003-04-25 2009-01-06 Medtronic, Inc. Optical detector for enzyme activation
US8003373B2 (en) 2003-04-25 2011-08-23 Medtronic, Inc. Optical detector for enzyme activation
US8940522B2 (en) 2003-04-25 2015-01-27 Medtronic, Inc. Optical detector for use in therapy
WO2011049431A1 (fr) * 2009-10-19 2011-04-28 Stichting Katholieke Universiteit, Radboud Universiteit Nijmegen Mesure du statut de coagulation sanguine dans la salive
WO2011049442A3 (fr) * 2009-10-19 2011-06-30 Stichting Katholieke Universiteit, Radboud Universiteit Nijmegen Mesure du statut de coagulation sanguine dans la salive
EP3422011A1 (fr) * 2017-06-28 2019-01-02 Koninklijke Philips N.V. L'estimation de valeur de paramètre dans un système de coagulation
WO2019002324A1 (fr) * 2017-06-28 2019-01-03 Koninklijke Philips N.V. Estimation de valeur de paramètre dans un système de coagulation

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