A quantitative thrombin time for determining levels of hirudin and Hirulog.
The anticoagulant effect of recombinant hirudin (rHir) and Hirulog has been monitored in patients with the activated partial thromboplastin time. Accurate monitoring with this test cannot be achieved if plasmas contain heparin, lupus anticoagulants, low concentrations of fibrinogen or other factors, or elevated fibrinogen-fibrin degradation products (FDP). We have therefore developed a simple, rapid, sensitive clot-based method, the quantitative thrombin time (QTT), to measure levels of rHir and Hirulog in patient plasma (or whole blood). The QTT is performed by mixing a 1:10 dilution of patient plasma (50 microliters) with human fibrinogen (50 microliters, 128 mg/dl) at 37 degrees C; the clotting time is initiated by adding human thrombin (50 microliters, 5-7.5 U/ml). The concentration of Hirulog or rHir in plasma can be determined by comparing the QTT in patient plasma with a standard curve that is generated by adding different concentrations of anticoagulant to pooled normal plasma. Studies with whole blood using the same procedure yield similar results. In the absence of Hirulog or rHir, the baseline QTT is the same in normal and abnormal plasmas (fibrinogen < 150 mg/dl and FDP as high as 1024 micrograms/ml, elevated FDP alone, lupus anticoagulant, or heparin < 0.9 U/ml). When known concentrations of either rHir or Hirulog are added to abnormal plasmas, the mean observed concentrations as determined by the QTT deviate from the expected values by less than 10% (range 0-19%). The data indicate that the QTT is a simple, rapid, and accurate test for the determination of levels of rHir and Hirulog in plasma or whole blood.
Reid
T J
TJ
Department of Hematology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
Alving
B M
BM
D006493:Heparin / Q000302:isolation & purification; D006629:Hirudins / Q000031:analogs & derivatives* / Q000097:blood*; D006801:Humans; D008991:Monitoring, Physiologic; D010314:Partial Thromboplastin Time; D010446:Peptide Fragments / Q000097:blood*; D011479:Protamines; D011994:Recombinant Proteins / Q000097:blood; D012015:Reference Standards; D013917:Thrombin / Q000037:antagonists & inhibitors*; D013918:Thrombin Time
D006629:Hirudins; D010446:Peptide Fragments; D011479:Protamines; D011994:Recombinant Proteins; D006493:Heparin; D013917:Thrombin; C074619:bivalirudin