Blood factor monitoring assay and uses thereof

Abstract

The present disclosure provides methods and compositions for diagnosing and treating subject having a bleeding disorder. The disclosed methods comprise contacting a sample, e.g., a blood or plasma sample obtained from the patient, with an activation mixture comprising an activated coagulation factor and a phospholipid mixture, wherein the activation mixture is dried onto a solid substrate. Also provided is a global hemostasis test based on the integration of clotting time (Ct) and pharmacokinetics data. The methods and compositions presented can be applied to point-of-care diagnostic systems.

1 . A composition for the measurement of coagulation factor activity in a sample comprising an activated coagulation factor and a phospholipid mixture, wherein the composition is dried onto a solid substrate. 
     
     
         2 - 4 . (canceled) 
     
     
         5 . The composition of any one of  claim 1 , wherein the coagulation factor is FVII, FVIII, FIX, or a fragment, variant, or derivative thereof. 
     
     
         6 - 13 . (canceled) 
     
     
         14 . The composition of  claim 1 , wherein the phospholipid mixture comprises phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, or combinations thereof. 
     
     
         15 . (canceled) 
     
     
         16 . The composition of  claim 1 , wherein the phospholipid mixture comprises 70 mole-% of phosphatidylcholine and 30 mole-% of phosphatidylserine. 
     
     
         17 . The composition of  claim 1 , wherein the phospholipid mixture comprises 80 mole-% of phosphatidylcholine, 10 mole-% of phosphatidylserine, and 10 mole-% of phosphatidylglycerol. 
     
     
         18 . The composition of  claim 1 , wherein the phospholipid mixture comprises 75 mole-% of phosphatidylcholine, 20 mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol. 
     
     
         19 . The composition of  claim 1 , wherein the phospholipid mixture further comprises cholesterol. 
     
     
         20 . The composition of  claim 19 , wherein the cholesterol content in the phospholipid mixture is from about 1 to about 20 mole-% of cholesterol. 
     
     
         21 - 28 . (canceled) 
     
     
         29 . The composition of  claim 1 , wherein the composition comprises 80% of 0.1 mg/mL Factor IXa and 20% of a phospholipid mixture comprising 75 mole-% of phosphatidylcholine, 20 mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol. 
     
     
         30 . The composition for the of  claim 1 , wherein the composition comprises 80% of a Factor XIa suspension at about 0.1-0.5 mg/mL and 20% of a phospholipid mixture comprising 75 mole-% of phosphatidylcholine, 20 mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol, wherein said composition is dried onto a solid substrate. 
     
     
         31 . A kit for performing a measurement of coagulation factor activity or coagulation time in a sample comprising the composition of  claim 1 . 
     
     
         32 - 43 . (canceled) 
     
     
         44 . A method for determining a coagulation factor level in a bleeding disorder patient, comprising:
 (a) contacting a sample obtained from the patient with an activation mixture comprising an activated coagulation factor and a phospholipid mixture, wherein the activation mixture is dried onto a solid substrate;   (b) measuring the time between the contacting of the activation mixture with the sample and the onset of clotting, thereby calculating the clotting time (Ct); and,   (c) correlating the Ct value with the level of coagulation factor in the sample.   
     
     
         45 . The method of  claim 44 , wherein the correlation between Ct and coagulation factor level (% Factor) is calculated according to the formula:
   Ct= A ×Ln(% Factor)+ B   [Formula I]
   
       wherein, for each coagulation factor, A is a constant value corresponding to the slope of a Ct versus coagulation factor concentration dose-response, and B is patient-specific off-set value. 
     
     
         46 - 83 . (canceled) 
     
     
         84 . The method of  claim 45 , wherein the phospholipid mixture comprises 70 mole-% of phosphatidylcholine and 30 mole-% of phosphatidylserine. 
     
     
         85 . The method of  claim 45 , wherein the phospholipid mixture comprises 80 mole-% of phosphatidylcholine, 10 mole-% of phosphatidylserine, and 10 mole-% of phosphatidylglycerol. 
     
     
         86 . The method of  claim 45 , wherein the phospholipid mixture comprises 75 mole-% of phosphatidylcholine, 20 mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol. 
     
     
         87 . The method of  claim 45 , wherein the phospholipid mixture further comprises cholesterol. 
     
     
         88 . The method of  claim 87 , wherein the cholesterol content in the phospholipid mixture is from about 1 to about 20 mole-% of cholesterol. 
     
     
         89 - 108 . (canceled)