[Study on Platelet Adhesion and Aggregation Induced by Gradient Shear Stress Using Microfluidic Chip Technology].

OBJECTIVE: To study the effect of gradient shear stress on platelet aggregation by microfluidic chip Technology. METHODS: Microfluidic chip was used to simulate 80% fixed stenotic microchannel, and the hydrodynamic behavior of the stenotic microchannel model was analyzed by the finite element analysis module of sollidwork software. Microfluidic chip was used to analyze the adhesion and aggregation behavior of platelets in patients with different diseases, and flow cytometry was used to detect expression of the platelet activation marker CD62p. Aspirin, Tirofiban and protocatechuic acid were used to treat the blood, and the adhesion and aggregation of platelets were observed by fluorescence microscope. RESULTS: The gradient fluid shear rate produced by the stenosis model of microfluidic chip could induce platelet aggregation, and the degree of platelet adhesion and aggregation increased with the increase of shear rate within a certain range of shear rate. The effect of platelet aggregation in patients with arterial thrombotic diseases were significantly higher than normal group (P<0.05), and the effect of platelet aggregation in patients with myelodysplastic disease was lower than normal group (P<0.05). CONCLUSION: The microfluidic chip analysis technology can accurately analyze and evaluate the platelet adhesion and aggregation effects of various thrombotic diseases unde the environment of the shear rate, and is helpful for auxiliary diagnosis of clinical thrombotic diseases.

[Performance Evaluation of a New Microfluidic Platelet Function Test Platform].

OBJECTIVE: To evaluate the performance of a microfluidic platelet function test platform (MPFTP) previously established by our research group. METHODS: The effects of flow shear rate and storage time on platelet function test were analyzed taking the MPFTP as the object. The intra-assay variability of the MPFTP was evaluated. The function of platelet in peripheral venous blood from 24 healthy volunteers was assessed using the MPFTP and light transmission turbidity, either in the presence of 20 µmol/L acetylsalicylic acid (AS, an inhibitor of cyclooxygenase 1) or 50 µmol/L 5-phospho-2-methylthioadenosine (2-MeSAMP, a P2Y12 receptor inhibitor). The diagnostic performance of both methods in assaying platelet function inhibition by AS and 2-MeSAMP was analyzed by using receiver operating characteristic (ROC) curve. RESULTS: Under the flow shear rate of 1 500 s-1, our MPFTP could dynamically monitor platelet adhesion and aggregation, as well as quantify platelet function. Platelet aggregation increased with the increase of flow shear rate, while sample storage at room temperature for up to 5 h did not affect results of platelet function test. The intra-assay variability coefficient of variation of the MPFTP was <15%. The area under the curve of ROC showed that this platform had good diagnostic performance in the identification of platelet function inhibition by AS and 2-MeSAMP. CONCLUSION: This MPFTP shows good analytical performance for the assay of platelet function and can be developed into a new clinical platelet function test device in the future.

[3D-Morphological Analysis of Inhibitory Effect of Aspirin on Platelet Aggregation].

OBJECTIVE: To develop a new method for evaluating the inhibitory effect of aspirin on platelet by the three-dimensional (3D) morphological parameters. METHODS: The sodium citrate-anticoagulant peripheral blood samples collected from 12 healthy volunteers were divided into 2 groups: group treated with 200 µmol/L acetylsalicylic acid (ASA), and control group. The platelets in the 2 groups were washed and purified. The purified platelets were added into reaction pools modified with type I collagen and induced to activation and aggregation under static condition. The 3D morphology of the formed platelet aggregate was measured by the laser shape microscopic system. Meanwhile, the platelet function was detected by turbidometric light transmittance aggregometry (LTA). RESULTS: This technology could acquire the shape, height and 3D morphology of the platelet aggregates without label, and could quantify their volume parameters. ASA treatment could obviously change the morphology of platelet aggregates. Compared with the parameters of control group, the volume of platelet aggregates in experimental group decreased significantly (t=8.97, P<0.01), while the cross-sectional area showed no significant change (t=1.94, P>0.05). The receiver-operating characteristic curve(ROC) analysis showed that the platelet aggregate volume as a parameter to identify the ASA inhibition effect had 91.7% sensitivity and 75% specificity when the cut-off value equal to 1395 µm3, and its accuracy and sensitivity were both better than those of platelet aggregates rate measured by LTA method. CONCLUSION: The new method developed for evaluating the ASA inhibition of platelet aggregation may provide a complementary strategy for researching and clinically evaluating of ASA anti-platelet aggregation in future.

[Dynamical Analysis of the Inhibitory Effect of Aspirin and Clopidogrel on Platelet Adhesion and Aggregation for Healthy People under Physiological Flow Condition by Microfluidic Chip Technology].

Objective To explore the inhibitory effect of aspirin and clopidogrel on platelet adhesion and aggregation behaviors under the physiological flow condition using microfluidic chip technology for health volunteers. Methods Peripheral venous blood samples collected from twelve randomly recruited health volunteers were treated with 20 µmol/L acetylsalicylic acid,50 µmol/L 2-methlthioadenosine-5'-monophosphate triethylammonium salt,and their combination,respectively,with untreated blood samples being control group. The blood samples were flowed through a microchannel modified with type I collagen protein at a physiological relevant shear rate of 1000 s-1 for 300 s,while the fluorescent images of platelet aggregations were dynamic captured using a microscope. Based on the images,the platelet coverage rates were calculated and used as quantitative parameters for evaluating platelet adhesion and aggregation behaviors. Results Under a flow condition of 1000 s-1 shear rate,an expected in vivo-like platelet adhesion and aggregation behaviors were observed at the surfaces of collagen proteins for control blood samples. Aspirin alone or clopidogrel alone suppressed platelet adhesion and aggregation at the later period of flow(200-300 s),while the combination of aspirin and clopidogrel reduced the adhesion numbers of platelets at the earlier stage of flow(≤150 s) and compromised the stability of platelet aggregation at the later period of flow(200-300 s). The combination showed synergistic effect in inhibiting platelet aggregation. Furthermore,such inhibitory effect was heterogeneous among 12 volunteers. Conclusion This simple microfluidic technology can offer a new technical platform for analyzing the inhibitory effect of antiplatelet drugs.