Resonant Acoustic Tweezing Spectroscopy for Small-Volume Noncontact Assessment of Blood Coagulation
Authors: Kasireddy Nithya , Orie Jeremy C , Khismatullin Damir 
Published: October 2019

Abstract
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.