Validation of a modified thromboelastometry approach to detect changes in fibrinolytic activity

Abstract

Background: Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans. The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay.

Methods: Blood was used of healthy volunteers, patients with sepsis, patients after cardiothoracic surgery, pregnant women, and cirrhotic liver disease patients. A whole blood tissue factor (TF) activated ROTEM assay with and without the addition of recombinant tissue plasminogen activator (rTPA) was developed. Plasma fibrinolysis determinants were measured in all volunteers and patients.

Results: Thirty five pM TF and additions of 125 and 175 ng/ml rTPA resulted in full lysis within 60 min in healthy volunteers. Coefficients of variation were below 10 % without and below 20 % with rTPA addition. In sepsis the hypofibrinolytic ROTEM profiles with 175 ng/ml rTPA were in line with the plasma determinants (high PAI-1, high fibrinogen, low tPA activity, and high d-dimers). After cardiothoracic surgery, reduced fibrinogen and platelet levels accounted for the reduced maximum clot firmness. The hypofibrinolytic profile is attributed to tranexamic acid use and elevated PAI-1 levels. The lowest rTPA concentration in cirrhosis resulted in hyperfibrinolysis in only few of the patients. In pregnancy normal profiles were found.

Discussion: Our high rTPA concentration demonstrates hypofibrinolytic profiles adequately in sepsis and after cardiothoracic surgery. Our low rTPA concentration of 125 ng/ml seems too high for demonstrating hyperfibrinolysis in cirrhotic liver disease.

Conclusions: We were able to present a validated whole blood ROTEM approach to fibrinolysis testing using added rTPA, which can be of added value next to classical plasma based fibrinolysis assays.

Keywords: Blood coagulation tests; Fibrinolysis; Thrombelastography; Tissue plasminogen activator; Validation studies.

Fig. 1

Whole blood stability when performing rTPA induced fibrinolysis on a ROTEM. Whole blood stability at room temperature (at 0,5 to 4 h after blood drawing) of 3 healthy volunteers for ROTEM parameters at 175 ng/mL rTPA; mean per time point (±SEM); overall mean (solid horizontal line on Y-axis) ± error margin based on CV of test (dotted horizontal lines on Y-axis); fitted line through all value points (grey declining line). CT clotting time, CV correlation of variation, MCF maximum clot firmness, LOT lysis onset time, LT lysis time

Fig. 2

Normal and rTPA induced fibrinolysis profiles on a ROTEM device. Median tissue factor stimulated ROTEM traces (coloured solid lines) with different rTPA concentrations expressed as interquartile ranges (coloured dotted lines) of four patient groups compared to healthy volunteers (black and grey). CTS-TXA cardiothoracic surgery patients using tranexamic acid, CTS-No TXA cardiothoracic surgery patient not using tranexamic acid, rTPA recombinant tissue plasminogen activator

Fig. 3

Boxplots of different ROTEM parameters at 125 ng/mL rTPA in healthy volunteers and patient groups. Sepsis and CTS are significantly different to HV (p < 0.001) for all four parameters. The grey area depicts the 95 % reference ranges as was calculated using the healthy volunteer data from Table 1. CTS-TXA cardiothoracic surgery patients using tranexamic acid, HV healthy volunteers, rTPA recombinant tissue plasminogen activator, CT clotting time, MCF maximum clot firmness, LOT lysis onset time, LT lysis time, FS fibrinolysis speed, defined as the decline in %/min between the LOT and LT points thus: FS = 75/(LT_min-LOT_min); FS_c, corrected fibrinolysis speed in mm/min between the LOT and LT points thus: FS_c = (amplitude_LOT-amplitude_LT)/(LT_min-LOT_min)

Fig. 4

Boxplots of different ROTEM parameters at 175 ng/mL rTPA in healthy volunteers and patient groups. Sepsis and CTS are significantly different to HV (p < 0.001) for all four parameters. The grey area depicts the 95 % reference ranges as was calculated using the healthy volunteer data from Table 1. CTS-TXA; cardiothoracic surgery patients using tranexamic acid; HV, healthy volunteers; rTPA, recombinant tissue plasminogen activator; CT, clotting time; MCF, maximum clot firmness; LOT, lysis onset time; LT, lysis time; FS, fibrinolysis speed, defined as the decline in %/min between the LOT and LT points; thus: FS = 75/(LT_min-LOT_min); FS_c, corrected fibrinolysis speed in mm/min between the LOT and LT points; thus: FS_c = (amplitude_LOT-amplitude_LT)/(LT_min-LOT_min)

Fig. 5

Boxplots of the fibrinolysis determinants and net effect (D-dimer) respectively, in healthy volunteers and patient groups. The grey areas depict the in house established reference range of fibrinogen and d-dimer levels, while they represent the reference ranges according to the insert of the manufacturer in the four other parameters. CTS-TXA cardiothoracic surgery patients using tranexamic acid, HV healthy volunteers, rTPA recombinant tissue plasminogen activator, PAI-1 plasminogen activator inhibitor, TAFI thrombin activated fibrinolysis inhibitor

Fig. 6

Boxplots of euglobulin clotlysis times in healthy volunteers and patient groups. The grey areas depict the reference range of euglobulin clotlysis time. CTS-TXA cardiothoracic surgery patients using tranexamic acid, ECT euglobulin clotlysis time, HV healthy volunteers