Kit for measuring the thrombin generation in a sample of a patient's blood or plasma

Abstract

The invention provides a kit for measuring the thrombin generation in a sample of a patient's blood or plasma, or in a sample of clotting factors. The kit contains lyophilized tissue factor/phospholipid-complex and a lyophilized mixture containing a thrombin-substrate and CaCl 2 . The invention also provides processes for preparing the reagents for the kit. The kit can be used in a method for measuring the thrombin generation in a sample, wherein it is possible to detect changes in thrombin generation kinetics, for example after administration of inhibitor bypassing agents to a patient who has developed inhibitors to an exogenous clotting factor such as Factor VIII.

1 . A kit for measuring the thrombin generation in a sample, comprising a lyophilized tissue factor (TF)/phospholipid (PL)-complex and a lyophilized mixture containing a thrombin-substrate and CaCl 2 .  
   
   
       2 . The kit according to  claim 1 , wherein the concentration of TF in the lyophilized TF/PL-complex ranges from about 5 to about 1000 pM.  
   
   
       3 . The kit according to  claim 1 , wherein the concentration of PL in the lyophilized TF/PL-complex ranges from about 1 to about 100 μM.  
   
   
       4 . The kit according to  claim 1 , wherein said TF or at least a functional part thereof is of natural or recombinant origin.  
   
   
       5 . The kit according to  claim 1 , wherein said PL is of natural or synthetic origin.  
   
   
       6 . The kit according to  claim 1 , wherein said PL is selected from the group consisting of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and mixtures thereof.  
   
   
       7 . The kit according to  claim 6 , wherein the weight ratio of PC/PS is in the range of from about 60/40 to about 95/5.  
   
   
       8 . The kit according to  claim 6 , wherein the weight ratio of PC/PS/PE is in the range of from about 60/20/20 to about 78/17/5, based on the total amount of phospholipids.  
   
   
       9 . The kit according to  claim 1 , wherein the thrombin-substrate contains a fluorescent or radioactive label.  
   
   
       10 . The kit according to  claim 1 , further comprising at least one thrombin standard.  
   
   
       11 . The kit according to  claim 1 , wherein the lyophilized TF/PL-complex is immobilized onto a support.  
   
   
       12 . The kit according to  claim 1 , wherein the lyophilized mixture containing a thrombin-substrate and CaCl 2  is immobilized onto a support.  
   
   
       13 . The kit according to  claim 11  or  12 , wherein the support is the inner surface of a vial or wells of an ELISA plate or strip.  
   
   
       14 . A process for preparing a lyophilized tissue factor (TF)/phospholipid (PL)-complex, comprising the following steps: 
 (a) preparing by extrusion phospholipid vesicles having a diameter in the range of about 200 to about 300 nm;    (b) lyophilizing the phospholipid vesicles to obtain a powder;    (c) reconstituting the powder with water for injection and mixing it with a tissue factor;    (d) freezing and thawing the mixture obtained in step (c) to form a TF/PL-complex;    (e) stabilizing the TF/PL-complex by incubating at about 4° C. for about 24 to 72 hours; and    (f) lyophilizing the TF/PL-complex.    
   
   
       15 . The process of  claim 14  wherein, between steps (e) and (f, the TF/PL-complex is diluted to an appropriate “ready to use” concentration.  
   
   
       16 . A process according to  claim 14 , wherein the TF/PL-complex in step (f) is immobilized onto a support by lyophilizing.  
   
   
       17 . A process according to  claim 16 , wherein the support is the inner surface of a vial or wells of an ELISA plate or strip.  
   
   
       18 . The process according to  claim 14 , wherein the TF/PL-complex in the step (f) is lyophilized without any preservatives.  
   
   
       19 . A process for preparing a lyophilized mixture containing a thrombin-substrate and CaCl 2 , comprising the following steps: 
 (a) dissolving a thrombin-substrate in a suitable solvent;    (b) adding CaCl 2  and dissolving the formed precipitate containing the thrombin-substrate and CaCl 2 ; and    (c) lyophilizing the mixture containing the thrombin-substrate and CaCl 2 .    
   
   
       20 . A process according to  claim 19 , wherein in step (c) the mixture is immobilized onto a support by lyophilizing.  
   
   
       21 . A process according to  claim 20 , wherein the support is the inner surface of a vial or wells of an ELISA plate or strip.  
   
   
       22 . A method for measuring the thrombin generation in a sample, comprising the steps of: 
 (a) providing a lyophilized tissue factor (TF)/phospholipid (PL)-complex and a lyophilized mixture containing thrombin-substrate and CaCl 2 ;    (b) contacting the sample with said lyophilized TF/PL-complex and said lyophilized mixture containing thrombin-substrate and CaCl 2 ; and    (c) measuring the thrombin generation in said sample.    
   
   
       23 . The method according to  claim 22 , wherein the sample is selected from the group consisting of whole blood, plasma and mixtures containing purified proteins from natural, synthetic or recombinant origin having haemostatic activity.