KIT FOR MEASURING THE THROMBIN GENERATION IN A SAMPLE OF A PATIENT'S BLOOD OR PLASMA
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 - 13 . (canceled)
14 . A method 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 method of claim 14 wherein, between steps (e) and (f), the TF/PL-complex is diluted to provide a concentration of TF that ranges from about 5 to about 1000 pM and a concentration of PL that ranges from about 1 to about 100 μM.
16 . The method of claim 14 , wherein the TF/PL-complex in step (f) is immobilized onto a support by lyophilizing.
17 . The to method of claim 16 , wherein the support is the inner surface of a vial or wells of an ELISA plate or strip.
18 . The to method of claim 14 , wherein the TF/PL-complex in step (f) is lyophilized without any preservatives.
19 . - 23 . (canceled)
24 . The method of claim 14 , wherein the concentration of TF in the TF-PL complex lyophilized in step (f) ranges from about 5 to about 1000 pM.
25 . The method of claim 14 , wherein the concentration of PL in the TF-PL complex lyophilized in step (f) ranges from about 1 to about 100 μM.
26 . The method of claim 14 , wherein the TF is of recombinant origin.
27 . The method of claim 14 , wherein the PL is a mixture comprising phosphatidylserine (PS) and phosphatidylcholine (PC); or a mixture comprising PS, PC, and phosphatidylethanolamine (PE).
28 . The method of claim 27 , wherein the PL is a mixture of PS and PC, and the weight ratio of PC/PS is in the range of from about 60/40 to about 95/5 based on the total amount of phospholipid.
29 . The method of claim 27 , wherein the PL is a mixture of PS, PC, and PE, and 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.
30 . The method of claim 14 , wherein step (d) comprises freezing the tissue factor with the phospholipid vesicles at about −20° C. overnight and thawing for about 30 minutes at room temperature.