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Knowledge of rheological properties, such as viscosity and elasticity, is necessary for efficient material processing and transportation as well as biological analysis. Existing rheometers operate with a large sample volume...

Introduction: Blood coagulation analysis is routinely performed to assess bleeding and thrombotic risks in surgical and critical care patients as well as in patients with diseases that cause coagulation abnormalities (e.g., hemophilia, thrombophilia and sickle cell disease). Majority of coagulation assays are based on photo-optical measurement of coagulation onset in blood plasma such as prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) and viscoelastic measurement of coagulating whole blood, often referred to as "global coagulation analysis", mostly done by thromboelastography (TEG, ROTEM) but they require large sample volume (> 0.5ml) requiring venipuncture, have poor standardization, and are unreliable tools to predict bleeding/thrombotic risk. Acoustic tweezing coagulometry (ATC) is an innovative noncontact drop-of-blood coagulation analysis technique that can perform both photo-optical and viscoelastic coagulation analysis with a sample volume as low as 4 μl to provide a comprehensive set of clinically relevant coagulation parameters such as blood viscosity, elasticity, reaction time, clotting rate, maximum clot stiffness, fibrin formation rate and cross-linking kinetics helpful for diagnosis and prediction of bleeding and thrombotic risks. ATC is particularly valuable for the pediatric patients as it enables safe and reliable point of care coagulation assessment with minimal sample volume. Materials and Methods: In this project, we demonstrate the feasibility of ATC for coagulation analysis by validation and standardization of the technique using whole blood collected from healthy adult volunteers and commercially purchased blood plasma. Further, we present the ability of ATC to assess bleeding risk in commercial blood plasma with coagulation FVIII deficiency with and without inhibitors, as well as whole blood collected from pediatric Hemophilia A patients without inhibitors. The time dependent changes in elasticity (elastic tweezograph, Figure 1A) and viscosity (viscous tweezograph, Figure 1B) of coagulating blood plasma or whole blood sample are used to extract the following coagulation parameters: clot initiation time (CIT), clotting rate (CR), clotting time (CT), time to firm clot formation (TFCF), and maximum clot stiffness (MCS) from elastic tweezograph; reaction time (RT), fibrin formation rate (FFR), and maximum fibrin level (MFL) from viscous tweezograph. Results and Discussion: Figure 1C shows the elastic tweezograph and figure 1D shows the viscous tweezograph of the healthy plasma, plasma with coagulation FVIII deficieny and plasma with inhibitors for coagulation FVIII activated via the intrinsic pathway of coagulation. The tweezographs suggest that the clot initiation is faster in healthy plasma compared to the FVIII deficient plasma and FVIII inhibitor plasma. The clotting rate is highest for healthy plasma followed by the FVIII deficient plasma and is the lowest for the plasma with FVIII inhibitors suggesting a delayed clot formation in the deficient and inhibitor groups. They all reach a similar final clot stiffness, but the time to firm clot formation is least in healthy plasma as expected and increases in the FVIII deficient group and further increases in the FVIII inhibitor group. Conclusions: Acoustic tweezing coagulometry can successfully measure the viscosity, elasticity and coagulation of whole blood and blood plasma with only a drop of the sample. This technique can successfully assess the bleeding risks in pediatric and adult patients with Hemophilia. Acknowledgements: This study has been supported by American Heart Association pre doctoral fellowship 20PRE35210991, U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559, PCT/US2018/014879 and PCT/US21/15336. Figure 1 Figure 1. Disclosures Kasireddy: Levisonics Inc.: Current Employment. Rafique: Pfizer Inc.: Consultancy; CSL Behring: Consultancy; HEMA Biologics: Consultancy. Khismatullin: Levisonics Inc.: Current equity holder in publicly-traded company; Levisonics Inc.: Patents & Royalties: PCT/US14/55559 (pending); Levisonics Inc.: Patents & Royalties: PCT/US2018/014879 (issued) ; Levisonics Inc.: Patents & Royalties: PCT/US21/15336 (pending)..

Acoustic tweezing rheometry is an innovative technology for low-volume non-contact rheological analysis of complex fluids characterized by increased sensitivity and accuracy as compared to traditional contact techniques. In this method, a small drop of a fluid sample is levitated in air by acoustic radiation forces and its viscoelasticity at different time instants is measured from drop shape changes. The acoustic tweezing rheometer operates in two different modes: quasi-static and oscillatory. This presentation focuses on the oscillatory technique in which the sample drop is forced into freely decaying shape oscillation by transient modulation of the standing acoustic field. Images of oscillating drops acquired by a high-speed camera are analyzed by a custom MATLAB code to obtain the shape amplitude vs. time curves and then the decay factor and resonance frequency of drop shape oscillation. Dynamic viscoelasticity of the sample (viscosity, relaxation time, and elastic modulus) was measured by applying the experimental data to the analytical formulae, derived from normal mode analysis of drop oscillation for viscoelastic fluid (Maxwell model) and viscoelastic solid (Kelvin-Voigt) materials. Using the oscillatory technique, we measured changes in blood viscoelasticity during coagulation and showed that the Kelvin-Voigt model leads to physically consistent results on rheology of coagulating blood.

Introduction: Measurement and interpretation of mechanical properties of whole blood and blood plasma are important diagnosis and treatment monitoring of various conditions like coagulopathy, hemophilia, sickle cell disease and many cardiovascular disorders. Many of the current techniques like thromboelastography, micro-viscometry or microfluidic devices used for this purpose require a large sample volume and/or may be prone to measurement errors due to sample contact with device walls. To address these issues, we developed a single-drop non-contact method for blood rheological analysis, referred to as "acoustic tweezing rheometry". With sample volume as small as 4 μL, our innovative technology has been successfully applied for assessment of whole blood and blood plasma coagulation. Here, we present the extension of this technology to resonant spectroscopic measurement of blood viscoelasticity. Materials and Methods: The schematic of the acoustic tweezing device is shown in (Figure 1A). The standing acoustic wave field between the transducer and reflector generates the acoustic radiation force on the biological sample that traps it in a host fluid (e.g., air). Sample tweezing (force-induced deformation and translational motion of the trapped sample) is achieved by amplitude modulation of the acoustic tweezing signal at high frequency and then decrease the frequency continuously until the lower limit for sample trapping is reached. During this frequency sweep, shape changes of the sample were recorded (Figure 1B) by a photodetector and a high-speed camera (Figure 1A). The amplitude-frequency response of the sample was obtained from raw data analysis, with the amplitude being the maximum deflection of the sample height from its equilibrium value. Dynamic (shear) viscosity and elasticity of the sample were assessed from the quality factor of the amplitude-frequency response (Figure 1C) and the resonance frequency, respectively. Results and Discussion: The quality factor analysis predicted that the dynamic viscosity of commercial normal control blood plasma was 1.5 mPa·s at room temperature, which agreed with previous large-sample-volume measurements. Once re-calcified, the resonance frequency of blood plasma and thus its shear elasticity increased due to clot formation until reaching a plateau in 5 min (Figure 1D). Using this graphical output (referred to as "tweezograph"), the following coagulation parameters can be extracted: clot initiation time, clotting rate, clotting time, and maximum clot elasticity. Conclusions: Resonant acoustic tweezing spectroscopy can accurately measure dynamic viscosity and elasticity of whole blood and blood plasma with a small drop of the sample and without artefacts or measurement errors due to sample contact with device walls. This technique can be applied for rapid assessment of whole blood and blood plasma coagulation. Acknowledgments: This study has been supported by U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559 and PCT/US2018/014879. Disclosures Khismatullin: Levisonics Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.