Thrombin generation in low plasma volumes

Abstract Accurate thrombin generation determination by calibrated automated thrombinography can be sustained when reducing the plasma and reagent volumes up to half, but not for higher reductions or plasma dilutions

Concomitant assessment of rivaroxaban concentration and its impact on thrombin generation

BACKGROUND: Reliable assays to measure direct oral anticoagulant (DOAC) levels and their activity in critical situations are needed. Drug levels alone are not representative of the effect of DOACs on an individual's coagulation. We developed a technique that provides direct assessment of the global effect of rivaroxaban on the individual's coagulation in addition to plasma concentrations. METHODS: DOAC concentrations were determined in fifty patients using rivaroxaban, with the new assay, Xross-CAT. The effect of rivaroxaban on coagulation (activity) was measured with thrombin generation (TG) in platelet poor plasma using 5 pM tissue factor on the same device. The levels were validated with the Biophen DiXal assay. The prothrombin time (PT) and dilute Russell viper venom time (dRVVT) were performed to estimate the effect on coagulation. RESULTS: The variability of Xross-CAT was below 12%. Xross-CAT correlates well with Biophen DiXaI (rs = 0.885). The bias, determined by Bland-Altman analysis, was 4.9% and the Passing-Bablok equation was y = 1.1x - 2.1. The correlation of plasma levels with TG was moderate (ETP rs = -0.548; Peak rs = -0.559), as for the PT (rs = 0.739) and the dRVVT (rs = 0.692). CONCLUSIONS: Xross-CAT shows a good correlation with Biophen DiXaI that was previously confirmed to accurately assess rivaroxaban levels. Bleeding and thrombotic complications are not necessarily associated with drug levels and could be influenced by concomitant risk factors. The main benefit of Xross-CAT is that it can be performed simultaneously with thrombin generation, providing an overview of the global anticoagulation status of a patient in relation to circulating DOAC levels

Perfusion bioreactors: a promising tool for the development of regulatory approvable bone tissue engineering constructs

In most bone fractures bone healing occurs spontaneously and leads to the repair of the defect. In critical size defects, however, a complete bone repair can often not be reached which leads to a permanent trauma. In these cases Tissue Engineered (TE) bone constructs (a scaffold combined with cells) can serve as a solution by implanting them at the side of the defect to further stimulate bone formation. To meet clinical standards, the European Medicines Agency (EMA) requirements related to safety, potency, efficacy and consistency need to be fulfilled. In this context perfusion bioreactors can become a valuable tool to control and monitor 3D cell growth in/on a scaffold in vitro in an efficient and consistent way. Our results indicate that perfusion bioreactors reduce the bone TE construct variability, and enable a real-time quality control by non-invasive characterization of cellular processes and/or environmental conditions. This makes them a valuable tool for the development of clinically approved Tissue Engineered bone constructs.status: publishe

Characterization and optimization of cell seeding in scaffolds by factorial design: quality by design approach for skeletal tissue engineering

Cell seeding into scaffolds plays a crucial role in the development of efficient bone tissue engineering constructs. Hence, it becomes imperative to identify the key factors that quantitatively predict reproducible and efficient seeding protocols. In this study, the optimization of a cell seeding process was investigated using design of experiments (DOE) statistical methods. Five seeding factors (cell type, scaffold type, seeding volume, seeding density, and seeding time) were selected and investigated by means of two response parameters, critically related to the cell seeding process: cell seeding efficiency (CSE) and cell-specific viability (CSV). In addition, cell spatial distribution (CSD) was analyzed by Live/Dead staining assays. Analysis identified a number of statistically significant main factor effects and interactions. Among the five seeding factors, only seeding volume and seeding time significantly affected CSE and CSV. Also, cell and scaffold type were involved in the interactions with other seeding factors. Within the investigated ranges, optimal conditions in terms of CSV and CSD were obtained when seeding cells in a regular scaffold with an excess of medium. The results of this case study contribute to a better understanding and definition of optimal process parameters for cell seeding. A DOE strategy can identify and optimize critical process variables to reduce the variability and assists in determining which variables should be carefully controlled during good manufacturing practice production to enable a clinically relevant implant.status: publishe

The anticoagulant effect of dabigatran is reflected in the lag time and time-to-peak, but not in the endogenous thrombin potential or peak, of thrombin generation.

Calibrated automated thrombinography (CAT) is a sensitive method to assess coagulation. Dabigatran inhibits both free thrombin and the αmacroglobulin (αM)-thrombin complex, which results in an erroneously increased peak and endogenous thrombin potential (ETP) without affecting lag time and time-to-peak. The aim of this study was to elucidate the artefacts in CAT when dabigatran is present. Thrombin generation (TG) was measured in vitro by using CAT in the presence or absence of 6 μM idarucizumab in plasma spiked with dabigatran. Additionally, ex vivo measurements were performed in plasmas of 63 patients using dabigatran in the presence and absence of idarucizumab. The in vitro experiments confirmed that the ETP, peak and velocity index were artificially increased. This was mainly due to the inhibition of the calibrator by dabigatran and partly due to CAT algorithms. The calibration artefact could be resolved by adding idarucizumab to the calibrator well. However, the second, mathematical artefact remains when dabigatran is present in the TG well. These findings were corroborated by ex vivo experiments i.e. the lag time and time-to-peak were significantly reduced in patients upon addition of idarucizumab, but the ETP and peak were not significantly affected. The velocity index did change significantly, since this is a combination of time-dependent factors and the peak. The peak, ETP and velocity index do not represent the anticoagulant effect of dabigatran on TG measured with CAT. The lag time and time-to-peak, however, do reflect the effect of dabigatran

Noninvasive Real-Time Monitoring by AlamarBlue (R) During In Vitro Culture of Three-Dimensional Tissue-Engineered Bone Constructs

Bone tissue engineering (TE) aims to develop reproducible and predictive three-dimensional (3D) TE constructs, defined as cell-seeded scaffolds produced by a controlled in vitro process, to heal or replace damaged and nonfunctional bone. To control and assure the quality of the bone TE constructs, a prerequisite for regulatory authorization, there is a need to develop noninvasive analysis techniques to evaluate TE constructs and to monitor their behavior in real time during in vitro culturing. Most analysis techniques, however, are limited to destructive end-point analyses. This study investigates the use of the nontoxic alamarBlue(®) (AB) reagent, which is an indicator for metabolic cell activity, for monitoring the cellularity of 3D TE constructs in vitro as part of a bioreactor culturing processes. Within the field of TE, bioreactors have a huge potential in the translation of TE concepts to the clinic. Hence, the use of the AB reagent was evaluated not only in static cultures, but also in dynamic cultures in a perfusion bioreactor setup. Hereto, the AB assay was successfully integrated in the bioreactor-driven TE construct culture process in a noninvasive way. The obtained results indicate a linear correlation between the overall metabolic activity and the total DNA content of a scaffold upon seeding as well as during the initial stages of cell proliferation. This makes the AB reagent a powerful tool to follow-up bone TE constructs in real-time during static as well as dynamic 3D cultures. Hence, the AB reagent can be successfully used to monitor and predict cell confluence in a growing 3D TE construct.status: publishe