PB1058 - Coag-Lysis assay, an automated and standardized combined turbidity plasma clot formation and lysis assay

Background: Clot properties are key in the development of thrombotic and bleeding complications. There is then an urgent need for a standardized clot turbidity and lysis assay to aid define healthy, hypo/hyper-coagulation and fibrinolysis status, i.e. the coagulation–fibrinolysis balance, facilitate interlaboratory data comparisons and improve patient’s care [Pieters JTH2018; Longstaff JTH2017].

Aims: Develop a fully automated Coag-Lysis Assay (CLA) for the measurement of the Coagulation-Fibrinolysis balance.

Methods: CLA was automated on a STA-R Max prototype using various concentrations of tissue factor (TF) (MP and PPP Reagents, Diagnostica Stago) with or without tissue Plasminogen Activator (h-single-chain tPA 0.10-0.30µg/mL) and calcium in undiluted plasma. FXIa (0-200pM) was added in the reagent for the Hemophilia A patients (HA). Heparin was inhibited by heparinase added in the reagent. Optical density deviation was recorded at 540nm during 10-30min at 37°C. Curve parameters were determined using a post-processing software. Experiments were performed in 58 healthy volunteers samples (HV); normal pool plasma spiked with DOACs or tinzaparin; plasma samples from VTE patients (VTE) and HA spiked with FVIII replacement therapy; and quality controls (QC) of hypo/hyper-coag/fibrinolysis.

Results: 0.13µg/mL tPA was determined to obtain lysis parameters within 30minutes. Addition of FXIa increased the assay sensitivity to low FVIII level. Within-run (n=21 measurements) and day-to-day (150 days) CVs determined using QC results were less than 6%-10% for coagulation and fibrinolysis. A 2SD normal range was established on HV plasmas.
Coagulation parameters were shorten with increasing TF concentrations, in hyper-coag QC or VTE plasma. CLA showed to be sensitive to DOAC concentration. Dabigatran enhanced clot susceptibility to fibrinolysis compared to other DOACs. Heparin neutralization using heparinase allowed CLA measurement in tinzaparin treated patients.

Conclusion(s): The fully automated CLA can be performed easily. It is available to assess bleeding or thrombotic tendency, based on plasma clot properties.