| Research purposes:The experiment of the preparation conditions of PRP is optimized.It is different from the traditional and the existing preparation method.Analyze the stability,economic and practical of PRP preparation from the concentrations of platelet and white blood cells,the preparation process of platelet activation and concentrations of growth factors.Research methods:The preparation of platelet rich plasma and platelets,leukocytes count:Take 46 m L of the anticoagulant whole blood from 20 cases included in the standard of healthy adult male volunteers respectively.After shaking,take 40 m L into 50 m L sterile centrifuge tube used in PRP preparation.Its two centrifugal conditions were 660 g/min centrifuge for 10 min.The final volume of PRP was about(6±0.2)m L.Blood cell analyzer was used to the determination of the platelet and white blood cells in whole blood and PRP.Platelet activity detection: Use flow cytometry to test CD42 b,CD62P and CD63(P-select element)on the surface of the platelets in the whole blood and PRP to analyze platelet activation before and after preparation.Growth factor detection: Use enzyme-linked immunosorbent assay(ELISA)to detect the concentrations of platelet derived growth factor-BB(PDGF-BB),transforming growth factor beta(TGF-?)and vascular endothelial growth factor(VEGF)secreted by platelets in activated whole blood,activated PRP and inactive PRP.And analyze the concentration of growth factors and their relationships with the number of platelets.The results of the study:The concentration analysis of platelets and leukocytes in whole blood and PRP : The concentrations of platelets in the whole blood and PRP respectively were(169.33+31.98)x109/L and(915.14+191.21)x109/L.Comparing the difference was statistically significant(t=20.384,P<0.01).The concentrations of leukocytes in the whole blood and PRP respectively were(6.24+1.36)x109/L and(18.86+6.73)x109/L.Comparing the difference was statistically significant(t=10.141,P<0.01).The concentrations of platelets and leukocytes in PRP positively correlate with that of platelets and white blood cells in whole blood.The correlation coefficients were r=0.884(P<0.01),r=0.877(P< 0.01)and were significantly positive correlation.Platelet activity detection: With streaming method by detecting CD42 b,CD63,CD62 p on platelet surface,calculate the enrichment coefficient of platelets respectively were 5.26± 0.68,6.03±1.09,5.22±0.66.The results were close to 5.41±0.54 tested by blood analyser.The method is verified further enrichment degree of platelets.The method was furtherly verified enrichment degree of platelets.In addition,what can be seen from the flow chart was that the expression rate of the platelet surface markers(CD62p and CD63 and CD42b)in whole blood and PRP was very close.It stated that there was no platelet activation in the process of preparation.The platelets maintained their original activity in PRP.Growth factor test results analysis: The testing results of the ELISA kit showed that the concentration of PDGF-BB,TGF-?1,VEGF after PRP activation were significantly different from the concentration in whole blood after activation(P < 0.01).The differences between PDGF-BB?TGF-?1?VEGF in inactive PRP and activated PRP had significantly statistical significance(P < 0.01).And statistical results showed these growth factors had no statistical significance between the inactive PRP and the activate whole blood(P > 0.05).Research conclusion:1.The PRP preparation method collects less anticoagulant whole blood,reducing the waste of the blood of patients;2.The PRP preparation method prepares PRP under the condition of room temperature without having a special instrument and reducing the economic burden of the patients.3.The PRP preparation method avoids the platelet activation in the process of centrifugal with the platelet original activity;4.This method can stably prepare PRP with high concentration of platelets and growth factors.This can well meet the clinical needs. |
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