# US 5,702,912 A · Method to determine the concentration of anticoagulants > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,702,912 A - Document ID: US5702912A - Status: Granted - Kind: A - Application number: 08/564,986 - Filed: 1995-11-30 - Granted: 1997-12-30 - Earliest claimed: 1990-11-05 - Assignees: Dade International Inc. - Inventors: Hendrick Coenraad Hemker; Robert Johan Wagenvoord; Hans-Jurgen Kolde - Web page: https://patentgrep.com/patent/US5702912A ## CPC - C12Q 1/56: involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen - G01N 2333/815: from leeches, e.g. hirudin, eglin - G01N 2333/974: Thrombin - G01N 2400/40: Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention relates to a method to determine the concentration of anticoagulants as a function of the inhibition of thrombin formation in an assay mixture. The assay mixture is composed of two reagents and the diluted plasma sample. The diluted plasma sample containing an anticoagulatory substance is mixed with a clotting factor reagent, which is a combination of an excess amount of purified coagulation factors of the endogenous system, phospholipids and a weak thrombin inhibitor, which can be potentiated by an anticoagulatory substance. Then a coagulation cascade activator and calcium chloride, an activator reagent, is added to the mixture and formed thrombin is measured after a chosen incubation time. A proportionality between the inhibition of the thrombin formation and the anticoagulatory active components allows for a determination of the concentration of anticoagulatory active components in the sample. ## Claims ### Claim 1 A method to determine a concentration of an anticoagulant in a sample as a function of thrombin formation in said sample comprising: (a) combining said sample with an amount of clotting factor reagent comprising prothrombin wherein the amount of clotting factor reagent is in excess of an amount of clotting factor present in a normal sample and is sufficient to initiate thrombin formation in said sample; (b) adding a sufficient amount of activator reagent to initiate coagulation; (c) adding a sufficient amount of a thrombin substrate to detect thrombin formation; (d) measuring thrombin formation; and (e) determining the amount of said anticoagulant in said sample by correlating the thrombin formation in said sample with thrombin formation in samples containing known amounts of said anticoagulant. ### Claim 2 (depends on 1) The method of claim 1 wherein said clotting factor reagent is comprised of clotting factor in excess, a sufficient amount of calcium ions to stabilize said clotting factor, a sufficient amount of phospholipids to catalyze the coagulation cascade reaction, and a sufficient amount of weak thrombin inhibitor to suppress thrombin formation in said sample. ### Claim 3 (depends on 2) The method of claim 2 wherein said anticoagulant is selected from the group consisting of: heparin, .alpha.-NAPAP and dermatan sulfate and the clotting factor reagent also comprises a sufficient amount of dextran sulfate to neutralize platelet factor 4 in said sample when the anticoagulant is heparin. ### Claim 4 (depends on 3) The method of claim 3 wherein said weak thrombin inhibitor is selected from the group consisting of antithrombin III, heparin cofactor II or a synthetic physiological inhibitor of thrombin which is potentiated by an anticoagulant. ### Claim 5 (depends on 1) The method of claim 1 wherein said clotting factor reagent is comprised of clotting factor in excess, a sufficient amount of calcium ions to stabilize said clotting factors, and a sufficient amount of phospholipids to catalyze the coagulation cascade reaction. ### Claim 6 (depends on 5) The method of claim 5 wherein said anticoagulant is selected from the group consisting of hirudin and synthetic physiological inhibitors of thrombin. ### Claim 7 (depends on 1) The method of claim 1 wherein the clotting factor reagent further comprises: Factor X, Factor VIII, Factor V, Factor IX or Factor IX-Factor XI complex. ### Claim 8 (depends on 1) The method of claim 1 wherein said activator reagent comprises a sufficient amount of Factor IXa, and CaCl.sub.2. ### Claim 9 (depends on 1) The method of claim 1 wherein said thrombin substrate is chromogenic, luminogenic, fluorogenic or electrogenic. ### Claim 10 (depends on 1) The method of claim 1 wherein said thrombin substrate is a fibrogenic solution. ### Claim 11 A method to determine a presence or concentration of an anti-coagulant in a sample, the method comprising: a) combining the sample with a clotting factor reagent comprising prothrombin wherein the prothrombin is added in an amount in excess of amounts present in normal samples, an activator of coagulation in an amount sufficient to initiate coagulation, and a thrombin substrate; b) measuring thrombin formation; and c) determining the presence or concentration of the anticoagulant by correlating thrombin formation of the sample with thrombin formation of samples containing known concentrations of said anti-coagulant. ### Claim 12 A kit to determine a concentration of an anticoagulant in a sample comprising: a. a first vessel containing a clotting factor reagent comprising prothrombin wherein clotting factors are in excess of a concentration of said clotting factors present in a normal sample; b. a second vessel containing an activator reagent; and c. a third vessel containing a thrombin substrate. ### Claim 13 A kit to determine a concentration of an anticoagulant in a sample comprising: a. a first vessel containing a clotting factor reagent comprising prothrombin wherein clotting factors are in excess of a concentration of said clotting factors present in a normal sample and a thrombin substrate; and b. a second vessel containing an activator reagent.