Deterioration in Recombinant Tissue Plasminogen Activator After Repeated Freezing and Thawing Cycles for Thromboelastography

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Background and Purpose: Thromboelastography (TEG) is often used to measure coagulation dynamics in the setting of acute ischemic stroke and thrombolytic therapy. The stability of thrombolytics has not been investigated in TEG. We conducted an experimental series to test the effects of recombinant tissue plasminogen activator (rtPA) on fibrinolysis in normal blood samples using TEG.

Methods: Freeze dried rtPA powder was reconstituted in normal saline containing 0.2% bovine serum albumin (100 mg/24 mL), divided into 1 mL aliquots, and diluted to enable using a relatively large volume for complete mixing with blood samples. Aliquots and dilutions were frozen at -20°C. The same rtPA dilution was thawed to ambient temperature before each use and refrozen until the next use over 4 testing days. Blood was drawn into 3.2% sodium citrated collection tubes. rtPA (100 μL) was added to 1 mL whole blood to achieve a 636 ng/mL rtPA TEG sample concentration. Control-whole blood and rtPA- whole blood TEG was performed for 3 h on 4 healthy human blood samples. Maximum clot amplitude (mm) and absolute clot strength (dynes/cm) were measured. Data (mean±SD) were analyzed by t-tests and significance inferred at p<0.05.

Results: Clot amplitude increased with thawing and refreezing (28±3, p=0.004; 35±2 p=0.01; 50±3, p=0.02; and 55±3, p=0.30; for testing cycles 1, 2, 3 and 4, respectively) compared to untreated samples (63±4). Clot strength also increased over the 4 cycles (2±0.3, p=0.007; 3±0.2, p=0.02; 5±05, p=0.01 and 6±0.7, p=0.30) compared to untreated (9±1.4). Lysis initiation time was gradually longer over the 4 tests (red arrows, Figure 1) suggesting delayed fibrinolysis.

Conclusions: One repeatedly thawed and refrozen rtPA stock showed a delay in fibrinolysis in healthy human blood, suggesting a loss of potency. Thus, rtPA should be aliquoted for 1-time use for experiments using TEG. Further investigation into rtPA potency deterioration with storage after reconstitution is warranted. (Figure presented).



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