Abstract
BackgroundThrombin generation (TG) assessed by Calibrated Automated Thrombogram (CAT-I) reflects the overall capacity of plasma to generate thrombin, thus evaluating the balance between the anti- and procoagulant processes. However, with this method the calibrator curve is usually not measured until completion which has a severe impact on the calculation of the TG parameters, especially under conditions where almost all substrate is consumed. In addition, direct thrombin inhibitor (DTI) cannot be present in the calibration sample due to inhibition of the calibrator. We have developed a modified TG assay (CAT-II) and performed head-to-head comparison with the CAT-I method using the same fluorometer. Furthermore, we have compared our CAT-II method to a new automated TG instrument (ST®-Genesia) using the same calibration method.MethodsTG was assessed with CAT-I and CAT-II using the same fulorometer and with ST®-Genesia in control plasma and plasma containing different anticoagulants (dabigatran, rivaroxaban, apixaban) and plasmas to which common interfering substances, bilirubin, hemoglobin and lipids were added. In CAT-I, calibration was against the same plasma containing calibrator in the presence of fluorogenic substrate (Z-GGR-AMC). In contrast, CAT-II method and ST®-Genesia used a standard concentration of thrombin in buffer and 7-amino-4-methylcoumarin (AMC) in a separate plasma sample for calibration.ResultsTG obtained from CAT-I using anticoagulant-free plasmas was lower compared with TG from CAT-II but both methods demonstrated an intra-assay variation less than 5% on all measured parameters. When comparing the two different calibration methods in the presence of different anticoagulants, a high correlation was seen in the presence of rivaroxaban and apixaban (R2 > 0.97), but not with dabigatran, a direct thrombin inhibitor. CAT-II method showed dose-dependent inhibition of TG in the presence of dabigatran, while CAT-I was not able to detect it. Both methods were able to correct for the interfering substances.ConclusionsOur results showed high similarity between the results of CAT-I and CAT-II method when it is applied in control plasmas and plasmas not inhibited with a direct thrombin inhibitor. Furthermore, both the CAT-II method and ST-Genesia using the same calibration method were able to detect the effect of all oral anticoagulants. Taken together, applying a new calibration method is a significant improvement for monitoring patients on direct thrombin inhibitors while not introducing any bias to results obtained on other types of samples.
Highlights
The Calibrated Automated Thrombogram (CAT-I) method was developed and designed for the measurement of thrombin generation in plasma [1]
Comparable intra-assay variation with Calibrated automated thrombogram (CAT)-I and CAT-II assays In order to compare the repeatability and precision of CAT-I and CAT-II assays, intra-assay coefficients of variation (CV%) were calculated using 7 thrombin generation curves derived from a non-treated plasma sample
All parameters derived from thrombin generation, e.g. lag time, endogenous thrombin potential (ETP), peak height and time to peak showed a between-run variation of less than 5% (Table 1)
Summary
The Calibrated Automated Thrombogram (CAT-I) method was developed and designed for the measurement of thrombin generation in plasma [1]. The same amount of AMC added to different plasma samples does not result in the same amount of fluorescent signal due to the variability of color between the different samples. Thrombin generation (TG) assessed by Calibrated Automated Thrombogram (CAT-I) reflects the overall capacity of plasma to generate thrombin, evaluating the balance between the anti- and procoagulant processes. With this method the calibrator curve is usually not measured until completion which has a severe impact on the calculation of the TG parameters, especially under conditions where almost all substrate is consumed. We have compared our CAT-II method to a new automated TG instrument (ST®-Genesia) using the same calibration method
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