Aims: Aim of the study was to show the suitability of the new coagulation analyzer Ceveron® alpha for determination of thrombin generation (TGA) in routine laboratories for discrimination of normal plasma samples from haemophilic and thrombophilic patient samples, from patients with a Lupus syndrome and patients with bleeding disorders.
Methods: TGA was determined by a thrombin generation assay based on monitoring the formation of thrombin by means of a fluorigenic substrate upon activation of the coagulation cascade by tissue factor and phospholipids using 2 reagents with different phospholipid concentrations (RClow and RChigh) on Ceveron® alpha. Thrombin generation is measured with a specially adapted TGA fluorimetric module which is placed over the cuvette rotor. Readout parameters were lag time (tLag), peak thrombin (Peak), time to peak(tPeak), velocity index (VI) and area under the curve (AUC). In case of patients with Lupus Anticoagulants a ratio of peak thrombin values triggered with RClow and RChigh was formed. Four laboratory groups participated in the study. All patient samples used for thrombin generation measurement were characterized by routine parameters.
Results: Group 1 analyzed samples with bleeding disorders. Peak thombin values showed the best discrimination between Hemophilia B patients (41.9, SD12.7nM), Hemophilia B patient after treatment (188.9nM) and normal controls (208.5nM, SD= 74.0). In Hemophilia A patients tLag was the best parameter to discriminate severe Hemophilia A (8.5min) from acquired hemophilia A and VWD (4.6 min, SD=2.1).
Normal ranges for TGA established in group 1 (n=16) were tLag 2.7min (SD=0.8), tPeak 7.8min (SD=1.9), peak thrombin 208.5nM (SD= 74), AUC 1769.4 (SD=327.9).
Group 2 analyzed samples of thrombophilic patients with pregnancy (n=30). Patient samples showed peak thrombin values (310.9nM, SD= 65.7) above normals (174.2nM, SD= 22.5). Even patients with heparin treatment had high peak thrombin values (340.5nM, SD=67.2). In heparin treated patients D-dimer values were pathologic as well.
Group 3 determined thrombin generation in patients with Lupus Anticoagulants determined by dRVVT, APTT-LA, Lupus-Ceveron and anti-cardiolipin antibodies (ACA). Patients only with high ACA values showed normal peak thrombin ratios of RClow/RChigh 0.6 (SD= 0.07) whereas the ratio in patients with high lupus anticoagulants as measured in the coagulation tests was 0.98 (SD= 0.31) for high antibody titer and 0.83 (SD 0.01) for low antibody titer. Inter-assay CVs in the normal control determined by this laboratory were 2.67% for peak thrombin and 4.83% for AUC and in thrombophilic plasma 3.0% for peak thrombin and 1.41% for AUC.
Group 4 determined TGA in orally anticoagulated patients. Normal range for peak thrombin established in this laboratory is 220nM (SD=52, n=12). Oral anticoagulant treatment lowered the AUC (from 1713.3nM, SD= 435.4 thrombin in samples with INR in the range 1 – 1.5 to 894.4nM, SD= 229.2 thrombin in samples with INR in the range 2 – 2.5) and peak thrombin formation (from 204.1nM, SD= 73.5 peak thrombin in samples with INR in the range 1 –1.5 to 79.0nM, SD= 35.2 peak thrombin in samples with INR in the range 2 – 2.5) and prolonged the tLag (from 5.5min, SD= 1.7 in samples with INR in the range 1 –1.5 to 7.6min, SD= 2.4 in samples with INR in the range 2 – 2.5.
Conclusions: TGA can be measured reproducibly with low variation under routine laboratory conditions. Peak thrombin is the best parameter for discrimination of patient samples from normals in haemophiliacs, in thrombophilia, patients with a Lupus syndrome, pregnancy and bleeding disorders. With automated, standardised thrombin generation measurement on the Ceveron® alpha large prospective studies can be conducted to confirm these results.
Disclosures: Wagner:Technoclone GmbH: Employment.