Activity levels of individual coagulation factors were evaluated using modified 1-step PT or aPTT methods. Clot formation in these tests was evaluated mechanically using an ST4 coagulation analyzer (Diagnostica Stago).
For FVII, a 1-step PT assay was used. Patient samples were diluted 1:10 in Owren Veronal buffer (Dade Bennng Siemens: Erlangen, Germany). 50 μl of factor-deficient plasma (Aniara, Westchester, Ohio) and 50 μL of diluted patient sample were warmed to 37°C in a cuvette with a metal mixing ball for 180 seconds. 100 μl of Dade Innovin PT reagent (Siemens Healthcare Diagnostics, Newark, Delaware) was added and the time to clot was recorded. Factor VII level was calculated in each sample relative to normal human plasma based on a standard curve.
For FIX and FX, a 2-step aPTT assay was used. Patient samples were diluted 1:20 in Owren Veronal buffer. 50 μl of diluted sample, 50 μL of aPTT reagent, and 50 μL of factor-deficient plasma (Aniara) were warmed to 37°C in a cuvette with a metal mixing ball for 300 seconds. 50 μl of CaCl2 was added and the time to clot was recorded. Factor IX and FX levels were calculated in each sample relative to normal human plasma.
For FVII, a 1-step PT assay was used. Patient samples were diluted 1:10 in Owren Veronal buffer (Dade Bennng Siemens: Erlangen, Germany). 50 μl of factor-deficient plasma (Aniara, Westchester, Ohio) and 50 μL of diluted patient sample were warmed to 37°C in a cuvette with a metal mixing ball for 180 seconds. 100 μl of Dade Innovin PT reagent (Siemens Healthcare Diagnostics, Newark, Delaware) was added and the time to clot was recorded. Factor VII level was calculated in each sample relative to normal human plasma based on a standard curve.
For FIX and FX, a 2-step aPTT assay was used. Patient samples were diluted 1:20 in Owren Veronal buffer. 50 μl of diluted sample, 50 μL of aPTT reagent, and 50 μL of factor-deficient plasma (Aniara) were warmed to 37°C in a cuvette with a metal mixing ball for 300 seconds. 50 μl of CaCl2 was added and the time to clot was recorded. Factor IX and FX levels were calculated in each sample relative to normal human plasma.