Assessment Of Hemostasis And Viability Of Red Blood Cells After Cryopreservation By Surface Acoustic Wave

Assessment of hemostasis,transfusion of red blood cells(RBCs)and fresh frozen plasma mixture are essential for anticoagulant therapy,traumatic surgery,cardiovascular diseases,etc.Coagulation test can predict whether there will be thrombosis or bleeding during the treatment.Meanwhile,transfusion of RBCs plays an important role in the prevention of coagulation disorders and clinical complications.Although the clinical values of conventional blood coagulation tests have been well demonstrated,these devices still have limitations,such as expensive equipment,excessive sample volumes,long turnaround times and difficult to miniaturization for point-of-care(POC)use.The existing POC equipment can only realize the routine coagulation test,and it is difficult to measure the hemodynamic parameters.In the process of clinical treatment,the RBCs are usually in the state of cryopreservation,and can be used after thawing and washing when necessary.However,the freeze-thaw-wash process will destruct the stability of RBCs,including the decrease of viscoelasticity,deformation,aggregation,and even hemolysis.Therefore,it is important to estimate the recovery(viability)of RBCs after thawing before use.The conventional method of RBCs recovery assessment,such as ultraviolet visible spectrophotometer(UVS),has no mechanical force interact with RBCs.As a result,it is unable to perceive the physical character such as viscoelasticity,deformation,aggregation,etc.,which can express the viability of RBCs.Aiming for these problems,this paper studies the characteristics of single-port Love mode resonant surface acoustic wave(SAW)and double-port Rayleigh wave delay line SAW(DRSAW).Two types of SAW devices were used respectively to evaluate the blood coagulation and recovery of RBCs after cryopreservation.The main works of the dissertation are as follows:1.Based on the theoretical analysis,it showed that the SAW sensor is sensitive to the mass and viscosity of the loaded samples.According to the viscoelastic characteristics of blood coagulation,the Maxwell viscoelastic model was established.Meanwhile,the influence of different parameters on the response of Love wave and Rayleigh wave SAW devices were further analyzed by finite element method(FEM)simulation.The simulation results for Love wave SAW device showed that the frequency response is approximately linear with the viscosity when the liquid to be measured in the low viscosity(Newtonian)region.With the increase of the viscosity,the frequency response trends to be saturated.2.Two types of SAW devices were fabricated according to the theoretical simulation results,including the selection of substrate materials,electrode materials and interdigital transducers(IDTs)design,as well as the methods of micro-and nanoscale fabrication.The single-port Love mode resonant SAW(SLSAW)was based on 64° YX-LiNbO3 substrate with a resonant frequency of 315 MHz and a guiding layer thickness of 4 μm.The DRSAW was based on 128° YX-LiNbO3 substrate with the operating frequency of 40 MHz.Resonant circuit,frequency measurement system and temperature control system were built up for SLSAW.The sensing signal of the SLSAW device was acquired by frequency and 64 Hz measurement accuracy of the frequency counter was achieved finally.For DRSAW,the circuit for measuring amplitude and phase difference was built up based on orthogonal phase sensitive detector.The measurement error of phase difference is less than 0.1° while the amplitude is less than 3 mV.3.Assessment of blood coagulation based on SLSAW was conducted.The designed SLSAW sensor works on harmonic resonance mode when blood samples transferred on it.Experiments show that the frequency shift of SLSAW ranges from approximately 8960 kHz to 10368 kHz,which indicates the enhancement of sensitivity and signal-to-noise ratio(SNR).To demonstrate the feasibility of the SLSAW,studies were carried out to examine the effects of clotting reagents on coagulation times and kinetics.Activated partial thromboplastin times of plasma were validated by comparing with SYSMEX CA-7000 with the correlation(R2)as 0.996.In terms of coagulation kinetics,reaction time,clot formation time,maximum frequency shift,and clot formation rate of whole blood,they were correlated well with corresponding parameters of standard TEG analyzer(R2=0.9942,0.9868,0.9712 and 0.9939,respectively).The results indicate that the SLSAW sensor can provide kinetic information that is beyond the scope of many POC coagulometers.SAW sensor with a relatively simple fabrication process and hundreds of megahertz operating frequency can be easily achieved.However,it can increase the challenge and cost to fabricate SAW sensor with much higher frequency and excellent performance.Therefore,harmonic resonate mode is an alternative to increase frequency response(sensitivity)with simple fabrication process and low cost.4.The recovery of cryopreserved RBCs was studied by the DRSAW.When 80%hematocrit RBCs and 0.9%NaCl solution were loaded on DRSAW respectively,the amplitude response of 80%hematocrit RBCs was greater than that of 0.9%NaCl solution,but the corresponding phase response changed irregularly.When the hemolytic RBCs and 0.9%NaCl solution were loaded on DRSAW respectively,both the amplitude and phase responses were not changed regularly.These indicate that the amplitude response can effectively distinguish 80%hematocrit RBC and 0.9%NaCl solution,but cannot perceive hemolytic RBCs.When the absorbance values of hemolytic RBCs obtained by different hemolytic methods were measured by UVS,remarkable differences could be observed,indicating that the measurement of recovery by UVS would be affected by different hemolytic methods.To verify whether the DRSAW has the potential to evaluate the recovery,fixed proportions of RBCs and hemolytic RBCs obtained by the same hemolysis method were tested by DRSAW and UVS,and the correlation coefficient R2 of recovery was 0.97283.Then the recovery of RBCs after cryopreservation was evaluated.During the evaluation of recovery of RBCs cryopreservation,5%low concentration glycerol was used as cryoprotectant,and different concentrations of sodium alginate(ALG)were used to encapsulate the RBC spheres,which were frozen after calcification.After thawing,the recovery was measured by UVS.The results showed that the recovery increased with the increase of ALG concentration.In the same cryopreservation method,the DRSAW was used to measure the recovery of RBCs after thawing.The correlation coefficient R2 between DRSAW and UVS was 0.99832.These results proved that DRSAW is reliable to evaluate the recovery of RBCs.