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Studying the mechanism of action of hemostatic agents using ElastoSens™ Bio

SUMMARY

The precise analysis of hemostatic agents (HA) in contact with blood are not possible with most conventional testing techniques.
ElastoSens™ Bio successfully measured the coagulation of blood in presence of HA through the clot viscoelastic properties.
It is shown here that increasing the concentration of HA in blood may affect the clot initiation time, clot strength and volume (swelling) depending on the mode of action (MoA) of the HA.
ElastoSens™ Bio can be used for R&D and development of HA, QC & manufacturing and for preclinical studies.

INTRODUCTION

Hemostatic agents (HA, e.g. powders, gauzes, adhesives and sealants) have been used for decades to control bleeding. The demand for these agents is growing due to two major trends in surgical practice: the expansion of minimally invasive surgery and complex reconstructive procedures that are more limited in their capacity to obtain hemostasis [1]. Hemostatic effectiveness (i.e. time to form a clot), clot swelling and mechanical strength of the formed clots are important parameters to be evaluated for bringing potential agents to the market. However, conventional techniques for analyzing HA such as visual inspection, thromboelastrography (TEG) and rotational thromboelastometry (ROTEM) can not precisely measure clotting kinetics, swelling, and the mechanical stability of the formed clots in the presence of hemostatic agents. In this short application note, two commercially available hemostatic agents, CELOX™ and QuikClot®, have been tested using the ElastoSens™ Bio to measure the viscoelasticity and swelling of the clots as a function of time during their formation.

MATERIALS AND METHODS

CELOX™ (MedTrade Products Ltd., Crewe, UK) and QuikClot® (Z-MEDICA, LLC, Wallingford, CT, USA) were added into the ElastoSens™ Bio sample holders at different powder/blood weight dosages: 0 %, 5 % and 10 % (w/w) for CELOX™ and 5 %, 10 % and 15 % (w/w) for QuikClot®. Sample holders and hemostatic agents were pre-heated at 37 °C into the thermal chamber of the instrument. Whole sheep blood in anticoagulants (Sodium Citrate) from Cedarlane (Burlington, ON, Canada) was heated at 37 °C in a water bath and then re-calcified by mixing with CaCl2. A volume of 5 mL of re-calcified blood was then pipetted and introduced into the sample holders containing the HA and the test was initiated. The test was performed at 37 °C during 40 minutes for CELOX™ and 20 minutes for QuikClot®. The ElastoSens™ Bio was used to measure in real time the shear storage modulus (G’) of the clot during its formation as well as the sample swelling ratio calculated as follows:

RESULTS AND DISCUSSION

Fig. 1 shows the time evolution of the shear storage modulus (A-C) and swelling ratio (B-D) of the blood with different concentrations of CELOX™ (0 %, 5 %, 10 %) and QuikClot® (5 %, 10 %, 15 %). The coagulation of re-calcified whole blood with no hemostatic agent provided a baseline (blood only control) to compare with the samples that contain hemostatic agents. The coagulation kinetics of blood with 0 % CELOX™ exhibited fibrinolysis (breaking down of fibrin network) after 1500 s. The clot initiation time for CELOX™ (A) decreased by increasing the concentration of the agent while it remained relatively stable for the QuikClot® (C) at different concentrations. The higher ratio of hemostatic agent to blood (w/w %) increased clot strength for both hemostatic agents. This ratio also affected the maximum rate of clot formation for CELOX™ but it did not seem to significantly change those of QuikClot® (Table 1). These two hemostatic agents did not yield changes in the sample height, suggesting no significant clot swelling.

Fig. 1: Shear storage modulus (G’, Pa) and swelling ratio (%) as function of time of two commercial hemostatic agents at different concentrations: CELOX™ at 0 %, 5 % and 10 % and QuikClot® at 5 %, 10 % and 15 %.

Table 1: Clot initiation time (s) and maximum rate of clot formation (Pa/s) for each HA at different concentrations.

The differences between each HA can be related to their Mode of Action (MoA). CELOX is a chitosan-based HA that physically blocks bleeding through the formation of a crosslinked gel with the different components of blood. QuikClot® is a clay-based HA that has been suggested to act by: (1) absorbing water at the site of bleeding which can rapidly concentrate platelets and clotting factors and by (2) inducing a chemical reaction that activates the intrinsic coagulation pathway and platelets, both promoting coagulation [2-5].

CONCLUSION

Increasing the amount of HA in blood decreased the clot initiation time for CELOX™ while it remained relatively stable for QuikClot®. The higher concentration of both HA increased clot strength while no significant clot swelling was observed during the study.

PERSPECTIVES

ElastoSens™ Bio can directly measure the evolution of viscoelasticity during clot formation with a technology that allows robust measurements and an easy interaction.
The viscoelastic properties during clot formation can be used to understand the Mode of Action (MoA) and optimize the formulation of hemostatic agents.

ElastoSens™ Bio can be used for:
- R&D and Product Development: to get superior quantitative data to better evaluate product prototypes and accelerate research.
- QC & Manufacturing: to improve quality control processes, strengthen documented traceability, optimize costs and qualify suppliers.
- Preclinical studies: to simulate in vivo conditions to better predict clinical outcomes.

REFERENCES

[1] Wheat, J. C., & Wolf, J. S. (2009). Advances in bioadhesives, tissue sealants, and hemostatic agents. Urologic Clinics, 36(2), 265-275.

[2] Li, J., Cao, W., Lv, X. X., Jiang, L., Li, Y. J., Li, W. Z., … & Li, X. Y. (2013). Zeolite-based hemostat QuikClot releases calcium into blood and promotes blood coagulation in vitro. Acta Pharmacologica Sinica, 34(3), 367-372.

[3] Khoshmohabat, H., Paydar, S., Kazemi, H. M., & Dalfardi, B. (2016). Overview of agents used for emergency hemostasis. Trauma monthly, 21(1).

How to measure the coagulation kinetics of blood plasma using ElastoSens™ Bio?

The human body has a natural mechanism to stop bleeding after an injury. Platelets migrate to the site of injury and start to form a soft blood clot. This activates other clotting factors in the bloodstream triggering a chain reaction to form a harder blood clot that will stay firmly in place.

Effect of hemostatic agents on the coagulation of blood : in vitro quantitative characterization using ElastoSens™ Bio

The ElastoSens™ Bio was used to analyze the effects of hemostatic agents (HAs) on blood coagulation. The technology identified HAs that can alter clotting independently of the body's natural processes. These findings can optimize current HAs and aid in developing new ones.

How do hemostatic agents work ?

Hemostatic agents (HAs) can be absorbable, biological, or synthetic. Absorbable HAs, like gelatin or oxidized cellulose, speed up clotting and are naturally absorbed by the body. Biological HAs include thrombin, fibrinogen, and platelets which are key to blood clotting. Synthetic HAs, such as polyethylene glycol, form strong sealant matrices. The choice of HA depends on the type of bleeding, tissue interaction, and patient's coagulation profile. Instruments like the ElastoSens™ Bio provide valuable data on HA efficacy by measuring blood absorption and coagulation kinetics.

An inorganic biomaterial with great hemostatic potential

Scientists from Dalhousie University, led by Dr. Mark Joseph Filiaggi, investigated the sodium polyphosphate (NaPP) polymer as a potential hemostatic agent. They tested six formulations of the biomaterial, with varying degrees of polymerization and types of divalent cations. The hemostatic potential of these formulations was evaluated using various blood clotting assays. The biomaterial was mixed with coagulation reagents and recalcified blood or plasma in a tube, which was then shaken to visually assess blood or plasma flow. The clotting time was noted as the time required to achieve no flow. Surgifoam®, a commercial hemostatic agent, was used as a control.

In addition to vaccines, the viscoelasticity of coagulating blood can help optimizing outcomes in COVID-19 patients

In a recent study published in the Critical Care Medicine Journal, researchers from Western Michigan University, the University of Texas, and healthcare-related offices across the United States investigated the relationship between COVID-19 and blood coagulation disorders. They found that analyzing the viscoelastic changes in coagulating blood can provide personalized information on a patient's coagulation state, offering valuable insights for treatment optimization. The study highlights the importance of a personalized patient-oriented approach due to the diverse clinical profiles observed in COVID-19 cases.

Blood coagulation analysis using ElastoSens™ Bio

The viscoelastic properties of coagulating blood can be correlated with several diseases and genetic conditions that affect the natural blood coagulation process including bleeding disorders, hemophilia, rare factor deficiencies, von Willebrand disease and platelet function disorders. Therefore, the evaluation of blood clot properties can be valuable for the study, diagnosis and eventually treatment of these diseases.