Method for diagnosis of thrombotic disorders

Abstract

The present invention provides an in vitro method useful for the diagnosis of a thrombotic disorder in a subject, having or at risk of having the disorder. Specifically, the disorder exemplified herein is associated with APC resistant Factor V and Va. The clotting time of a test sample is analyzed in the presence and absence of APC and compared with a standard reference sample in order to diagnose the subject.

1 . An in vitro method for diagnosing a subject as having or as being at risk for having a thrombotic disorder associated with activated protein C (APC)-resistant factor V or Va, the method comprising: 
 a) contacting a test sample comprising a coagulation factor V or Va-containing specimen from the subject with a procoagulant reagent, factor V-deficient plasma to provide coagulation factors other than factors V or Va, calcium sufficient to initiate clotting, and APC in a test reaction; and    b) comparing the clotting time for the test reaction to the clotting time for a control reaction carried out under the same conditions as the test reaction, but with a control sample comprising a coagulation factor V or Va-containing specimen from an individual not having or not at risk of having a thrombotic disorder associated with APC-resistant factor V or Va, wherein: 
 i) detection of a decreased clotting time in the test reaction relative to the control reaction indicates a diagnosis of a thrombotic disorder associated with APC-resistant factor V or Va; and  
 ii) detection of a similar clotting time in the test reaction relative to the control reaction indicates that the subject does not have or is not at risk of developing a thrombotic disorder associated with APC-resistant factor V or Va.  
   
   
   
       2 . An in vitro method for diagnosing a subject as having or as being at risk for having a thrombotic disorder associated with activated protein C (APC)-resistant factor V or Va, wherein the subject is presently on an oral anticoagulant regimen, the method comprising: 
 a) contacting a test sample comprising a coagulation factor V or Va-containing specimen from the subject with a procoagulant reagent, factor V-deficient plasma to provide coagulation factors other than factors V or Va, calcium present in a concentration from about 5 mM to 15 mM, and APC present at from about 100 ng/ml to 10 μg/ml in a test reaction; and    b) comparing the clotting time for the test reaction to the clotting time for a control reaction carried out under the same conditions as the test reaction, but with a control sample comprising a coagulation factor V or Va-containing specimen from an individual not having or not at risk of having a thrombotic disorder associated with APC-resistant factor V or Va, wherein: 
 i) detection of a decreased clotting time in the test reaction relative to the control reaction indicates a diagnosis of a thrombotic disorder associated with APC-resistant factor V or Va; and  
 ii) detection of a similar clotting time in the test reaction relative to the control reaction indicates that the subject does not have or is not at risk of developing a thrombotic disorder associated with APC-resistant factor V or Va.  
   
   
   
       3 . The method of  claim 2 , wherein the specimen from the subject is previously frozen plasma.  
   
   
       4 . The method of  claim 2 , wherein the thrombotic disorder is thrombophilia.  
   
   
       5 . The method of  claim 2 , wherein the thrombotic disorder is due to a factor V mutation.  
   
   
       6 . The method of  claim 5 , wherein the mutation results in a change from arginine to glutamine at position 506 of factor V.  
   
   
       7 . The method of  claim 2 , wherein the procoagulant reagent comprises tissue factor.  
   
   
       8 . The method of  claim 2 , wherein the procoagulant reagent comprises a phospholipid.  
   
   
       9 . The method of  claim 8 , wherein the phospholipid is present at a concentration of about 5-100 μM in the test reaction.  
   
   
       10 . The method of  claim 8 , wherein the phospholipid is present at a concentration of about 10-50 μM in the test reaction.  
   
   
       11 . The method of  claim 2 , wherein the procoagulant reagent comprises an activator of the intrinsic coagulation pathway.  
   
   
       12 . The method of  claim 11 , wherein the activator is a clotting factor selected from the group consisting of factor Xa, factor IXa, factor XIa and factor XIIa.  
   
   
       13 . The method of  claim 2 , wherein the procoagulant is a reagent selected from the group consisting of kallikrein, Russell's viper venom, micronized silica particles, ellagic acid, sulfatides, kaolin, and tissue thromboplastin.  
   
   
       14 . The method of  claim 2  wherein the specimen from the subject is diluted in a physiologically balanced buffer.  
   
   
       15 . The method of  claim 2 , wherein the APC in the test reaction is present at from about 200 ng/ml to 1 μg/ml.  
   
   
       16 . The method of  claim 2 , wherein the anticoagulant is heparin.  
   
   
       17 . The method of  claim 2 , further comprising setting up a no-APC test reaction, carried out under the same conditions as the test reaction except that no APC is added to the reaction, wherein a clotting time for the test reaction that is similar to, or faster than, a clotting time for the no-APC reaction is indicative of a subject having a thrombotic disorder associated with a homozygous mutation from arginine to glutamine at position 506 of factor V.  
   
   
       18 . An in vitro method for diagnosing a subject as having or as being at risk for having a thrombotic disorder associated with activated protein C (APC)-resistant factor V or Va, wherein the subject is presently on an oral anticoagulant regimen, the method comprising: 
 a) contacting a test sample comprising a coagulation factor V or Va-containing specimen from the subject with a procoagulant reagent, factor V-deficient plasma to provide coagulation factors other than factors V or Va, calcium sufficient to initiate clotting, and APC; and    b) comparing the clotting time for the test reaction to the clotting time for a no-APC control reaction carried out under the same conditions as the test reaction, but without adding APC, wherein detection of a similar clotting time or faster clotting time in the test reaction relative to the no-APC control reaction indicates a diagnosis of a thrombotic disorder associated with APC-resistant factor V or Va.    
   
   
       19 . The method of  claim 18 , wherein a clotting time for the test reaction that is faster than, a clotting time for the no-APC reaction is indicative of a subject having a thrombotic disorder associated with a homozygous mutation in factor V or Va rendering the factor APC-resistant.  
   
   
       20 . The method of  claim 19 , wherein the mutation occurs at position 506 of factor V.  
   
   
       21 . The method of  claim 20 , wherein the mutation is a change from arginine to glutamine at position 506 of factor V.  
   
   
       22 . The method of  claim 2 , wherein an anticoagulant neutralization step is not performed before performing the method.