论文部分内容阅读
目的:通过分析髋关节骨折患者高凝状态与其外周血中微粒（microparticles，MPs）水平的相关性，探讨清除MPs纠正髋关节骨折患者中高凝状态的可行性。方法:2018年12月至2019年9月65例我院骨科急诊收治髋关节骨折患者资料，男24例，女41例；年龄（75.6±9.8）岁（范围：58~95岁）。其中股骨颈骨折27例，转子间骨折38例，术前均摄X线片或行CT扫描确诊。同期健康体检中心健康人群20例作为对照，男8例，女12例；年龄（72.3±6.5）岁（范围：56~81岁）。实验及对照组均在清晨空腹下取2 ml抗凝全血，采用透射电镜和纳米颗粒跟踪分析仪对全血中提纯的微粒进行鉴定；全血差速离心获得无细胞血浆（cell free plasma，CFP）后采用流式细胞仪测定其中膜联蛋白-5（annexin V，AV）阳性MPs和来源于血小板AVn +微粒（platelet-derived microparticles，PMPs）的数量及应用凝血和血小板功能分析仪测定凝血酶生成时间（activated clotting time，ACT）评价高凝状态程度，对与高凝状态相关的危险因素进行Logistic回归分析，判断测定MPs水平是否为高凝状态的独立危险因素，并进行相关性分析；最后应用全自动凝血分析仪测定各样本CFP在过滤去除MPs前后的凝血功能。n 结果:电镜下MPs形态呈现囊泡状，具有完整的双层膜结构，大小在100~1 000 nm范围内，并且形态不均一，有不规则囊泡状和圆形的囊泡状的大体形态，纳米颗粒跟踪分析仪可以看到髋关节周围骨折患者外周血中MPs大小为（239.7±4.0）nm；对照组MPs分布大小为（247.7±3.3）nm，且二者MPs直径大小差异无统计学意义（n P>0.05）。髋关节骨折患者循环AVn +MPs计数的平均水平为（564±171）个/μl，PMPs平均（326±104）个/μl，正常人群AVn +MPs计数的平均水平为（252±82）个/μl，PMPs平均（192±41）个/μl，两者AVn +MPs，PMPs之间的差异均有统计学意义（n P<0.05）；髋关节骨折患者ACT值平均水平为（324±94）s，而正常人群ACT平均水平为（535±76）s，二者之间的差异有统计学意义（n P<0.05）；单因素Logistic回归分析显示APTT、PMPs、AVn +MPs的水平可能是高凝状态的危险因素，将其进行多因素Logistic回归分析结果显示，AVn +MPs是高凝状态的独立危险因素。其与ACT间存在高度负相关（n r=-0.822，n P0.05）。n 结论:髋关节骨折患者存在高凝状态，且AV+MPs水平是高凝状态的独立危险因素，和ACT之间具有高度相关性，并且MPs对经典的凝血功能无明显影响。“,”Objective:To study the correlation between hypercoagulant status of patients with hip fracture and the level of microparticles (MPs) in their peripheral blood, and to explore the feasibility of removing MPs to correct hypercoagulant status in patients with hip fracture.Methods:Sixty-five patients from December 2018 to September 2019 with hip fracture were included. There were 24 males and 41 females with the average age of 75.6±9.8 years old (range 58-95 years). Among them, 27patients (43.1%) were femoral neck fracture and38 patients (56.9%) were intertrochanteric fracture.All patients were diagnosed with X-ray and CT. Meanwhile, about 20 healthy people in the physical examination center included as controls in our study. There were 8 males and 12 females with the average age of 72.3±6.5 years old (range 56-81years). 2 ml of anticoagulant whole blood was taken on an empty body in the morning, and purified microparticlesby whole-blood density gradient centrifugation in whole blood were identified by electron microscope and nanoparticle tracking analyzer. After cell free plasma (CFP) was obtained by whole-blood density gradient centrifugation, the number of whole annexin V (AV) positived MPs and these MPs which from platelet (PMPs) was determined by flow cytometry. The activated clotting time (ACT) was determined by coagulation and platelet function analyzer to evaluate the degree of hypercoagulability. Then, Logistic analysis was performed on risk factors associated with hypercoagulability to determine whether the level of MPs was an independent risk factor for hypercoagulability, and the correlation between ACT value and MPs level was analyzed. Finally, the four coagulation items of each sample CFP before and after MPs removal were determined by automatic coagulation analyzer.Results:Under electron microscopy, MPs presented vesicular appearance,with a complete double-layer membrane structure, the size was in the range of 100-1 000 nm, and the morphology was not uniform. there were irregular vesicular and circular vesicular general shapes. The average size of MPs in peripheral blood of patients with hip fractures was 239.7±4.0 nm. The mean size of MPs distribution in the control group was 247.7±3.3 nm, and there was no statistically significant difference in MPs diameter between the two groups. The average level of circulating AVn +MPs in patients with hip fracture was 564±171/μl, and the average level of PMPs was 326±104/μl. In the control group, the average level of AVn +MPs was 252±82/μl, the average level of PMPs was 192±41/μl, the difference between AVn +MPs and PMPs was statistically significant (n P<0.05). The average ACT level of patients with hip fracture was 324±94 s, while the average ACT level of the normal population was 535±76 s, and the difference between the two was statistically significant (n P<0.05). Single factor logistic regression analysis showed that the levels of APTT, PMPs and AVn +MPs may be risk factors for hypercoagulability, and multivariate logistic regression analysis showed that AVn +MPs is an independent risk factor for hypercoagulability.It has a highly negative correlation with ACT (n r=-0.822, n P<0.05). The results of four coagulation items determined by CFP were PT 10.8±0.46 s, APTT 30.6±1.56 s, Fib 3.08±0.36 g/L, INR 0.98±0.04 and TT 19.3±0.62 s. After the removal of MPs, the coagulation function was PT 10.8±0.52 s, APTT 32.4±3.0 s, Fib 2.90±0.33 g/L, INR 0.99±0.05 s, and TT 19.9±0.63 s. There was no statistically significant difference before and after coagulation function.n Conclusion:There is a hypercoagulable state in patients with hip fracture, moreover, the level of AVn +MPs is an independent risk factor for hypercoagulability, which is highly correlated with ACT, and MPs has no significant effect on the classic four factors of coagulation.n