Caracena, Thomas

Introduction: Previous studies have successfully created blood clot analogues for In-vitro testing using animal blood. Blood components vary greatly among species and thus, creating clot analogues with human blood is likely a more accurate representation of thrombi formed in the human vasculature. We present a novel method of creating clot analogues from human blood and platelets that mimic the process by which clots form In-vivo . Methods: Following IRB approval from Mayo Clinic, human whole blood and platelets donations were obtained from the Blood Transfusion service. The whole blood was centrifuged at 1,200RPM for 20 minutes to separate it into its constituents. Plasma was removed and the remaining Red Blood Cells and Buffy Coat were mixed together by inverting. A total of 12 clot analogues were created with varying concentrations of components; Red Blood cells/Buffy Coat, Plasma and Platelets. Thrombin was added first to stimulate platelets activation for a total of 5 mins whilst continuously mixing by inversion. The RBC/WBC mixture was added next followed by CaCl2. The mixture was then quickly drawn into a 3cc syringe and spun overnight at 20RPM at room temperature to mimic dynamic flow conditions. Macro-photographs were taken to display the variation in texture and color between different clot analogue types. The clots were then fixed in 10% neutral buffered formalin for 24 hours prior to being processed. Histopathological analysis was performed using Hematoxylin and Eosin (H&E) and Martius Scarlet Blue (MSB) staining to confirm clot composition. Results: Red Blood cell-rich, Fibrin-rich, Platelet-rich and mixed clot analogues that accurately mimic clots retrieved from Acute Ischemic Stroke Patients were created. The range of histopathological compositions of the clot analogues is similar to that of the clinical samples. Conclusions: The addition and activation of platelets is key to creating accurate clot analogues for In-vitro testing. Spinning the clots is important to prevent natural sedimentation and mimic the In-vivo situation.

Ischemic stroke results from thrombus becoming lodged in a vessel in the cerebrovascular circulation. The constituents of the thrombus can vary widely and have direct effects on the mechanical properties of the thrombus. Thrombectomy procedures could potentially be improved if the mechanical properties of the thrombus could be determined prior to treatment. In the study, we developed a new non-contact method associated with Rayleigh wave propagations for evaluating mechanical properties of human clotted blood, referred to as ultrasound-mediated optical coherence elastography (UOCE). Due to clot mechanical properties being associated with fibrin and platelet content, eight different clot analogues with varying fibrin/platelets concentrations, from 0.6% (control group) to 19.0%, were produced, and the clot analog in each concentration was repeatedly measured five times. A highly focused transducer with a center frequency of 7.5 MHz was used to generate acoustic radiation forces exerted on the surface of clotted blood to induce Rayleigh wave propagations. The transducer was driven by a 50 dB radio frequency amplifier a 4 ms pulse which was repeated at a rate of 19 Hz. The optical coherence tomography (OCT) acquisition was set to 50 lateral positions and 4000 A-scans for each lateral position at a 76 kHz scan rate for total acquisition time of 52.6 ms. The results demonstrated that the Rayleigh wave group velocities positively correlated with the concentrations of fibrin/platelets. The group velocities ranged from 0.78 ± 0.02 m/s for 0.6% fibrin/platelets and increased to 2.42 ± 0.06 m/s at a fibrin/platelets concentration of 19.0%. There are statistically significant difference ( p <0.05) between control group and all clot analogues with a fibrin/platelets concentration of greater than 2.3%. In conclusion, we propose UOCE as a novel approach capable of evaluating mechanical properties of human blood clots based on Rayleigh wave propagation. This sensitive method can be used for the evaluation of the mechanical properties of human blood clot analogues and thrombectomy samples.