Research On Time Estimation Of Deep Vein Thrombosis

Objective: Deep venous thrombosis is a venous reflux obstacle in deep vein caused by abnormal blood coagulation. And the sloughed thrombus can lead to pulmonary embolism. Since the formative process, pathogenesis and treatment of the two diseases are similar, we often name them as venous thromboembolism(VTE). VTE is the third vascular disease with incidence just after acute coronary syndrome and stroke. The statistics indicate that the fatal cases caused by thromboembolism encountered in forensic accounts for about 5%. In clinical medicine, pulmonary embolism is usually diagnosed depending on the hematology and imaging. While in forensic cases, the forensic pathologists usually combine the conventional forensic pathology inspection technology with autopsy to make appraisal. But VTE often occur in the conditions of trauma, active restriction and so on, the causality between trauma and pulmonary embolism needs to be clarified. And in the cases involving more than one risk factor, the questions about degree of involvement of each factor need to be clarified, such as time of thrombosis, time sequencing of the trauma and thrombus, the source of thrombus and so on. Nowadays the systemic study about the time estimation of thrombosis and the effective index and uniform standard in forensic cases is scanty. So the research on the time estimation of thrombosis has practical meaning in solving the forensic problem above.The experiment observed the histopathological change of position of thrombosis in rats' inferior vena cava using HE staining, Von Kossa staining, Perl's staining, Masson staining and tested the expression of glycoproteins-?a(CD61), CD34 and smooth muscle protein(alpha SMA) in platelet membrane in different time groups using immunohistochemistry staining on the basis of establishment of animal model of thrombosis in rats' inferior vena cava and in combination with the forensic cases to provide scientific evidence for time estimation of thrombosis in forensic cases.Methods:1 The animals experiments: HE staining, Von Kossa staining, Perl's staining, Masson staining and immunohistochemical staining of thrombus in rats' inferior vena cava. Experimental groups: 80 healthy clean(Sprague-Dawley, SD) male rats with the weight of 250g±10g were fed to adapt to the environment. The rats were divided into 10 groups including 0h, 3h, 6h, 12 h, 1d, 3d, 1w, 2w, 3w and 4w after stenosis operation of inferior vena cava. The rats in different groups were anesthetized to get inferior vena cava after the animal model was built. The materials were fixed in 10% neutral formalin. HE staining, Von Kossa staining, Perl's staining and Masson staining were performed in the vascular position of thrombosis. And the proteinic expression changes of CD61, CD34 and ?-SMA in different time groups were observed in the process of thrombosis using immunohistochemistry.2 Experiments of tissue specimens from human body: HE staining,immunohistochemical staining of CD61, CD34 and ?-SMA were performed on the formalin-fixed paraffin-embedding vessel of inferior deep vein and pulmonary artery with thrombosis after retrospective analysis of fatal cases resulted from PE in forensic identification center of Hebei Medical University to observe the pathological performance and protein expression and to analyze the relation between time of thrombosis and time of trauma formation.The Data were presented as "Mean±standard deviation(Mean±SD)", statistical analysis was carried out with SPSS 21.0 software. One-factor analysis of variance(ANOVA) and the least significant difference(LSD) were performed to compare mean of each group. A level of P < 0.05 was supposed to be statistically significant.Results:1 Animal experiments1.1 Thrombus staining of inferior vena cava1.1.1 HE staining of thrombus Intravascular congestion was observed in 0 hour group. In 3 and 6 hours group, lateral white thrombus formed in the interior of vessel and extended to the vascular lumen, some matters like cellulose separated out from the thrombus and neutrophils began to increase. Numerous platelet small beam formed and gathered in 12 hours group until the peak in 1day group, the cracks of platelet small beam was full of red and white blood cells. In 1-3 weeks group, fibroblasts were observed in the interface of thrombus and vessel wall in 3 days group. Endothelial cells appeared on the surface of crannies in the interior of thrombus resulting from contraction of thrombus to form new capillaries, the thrombus tightly attached to the vascular wall began to organize and numerous fibroblasts grew from adhesive position to the interior of thrombus, the nucleolus of monocyte increased, hemosiderin excluded, the bulk and number of macrophagocyte increased, calcium salt formed, some new white thrombus was observed in the vessel with second stenosis. Intimal hyperplasia occurred to reach repatency of vessel in 4 weeks group.1.1.2 Perl's staining of thrombus in inferior vena cava The hemosiderin particles can be dyed blue in Perl's staining. We found that hemosiderin particles scattered in the interior of thrombus could be first found in 3 days group of this experiment. The content of hemosiderin located in the edge of thrombus attached to the vascular wall increased rapidly and the hemosiderin was also could found in the interior of thrombus in 1-3 weeks groups. The difference of the 3 groups was not significant. Nouermous hemosiderin deposited in the organized thrombus in 4weeks group, and the difference with other groups was significant(P <0.05).1.1.3 Von Kossa staining of thrombus in inferior vena cava Calcium salt deposition can be dyed black in Von Kossa staining. Calcium salt deposition scattered on the edge of the thrombus in 1 and 2 weeks groups with no significant difference between the two groups. 3 and 4 week groups, the bulk and number of macrophagocytes containing calcium salt increased rapidly, the calcium salt without absorbing deposited in the thrombus to form phlebolith. The calcium salt deposition had a increasing tendency with time expanding. The difference of 3 and 4 weeks groups compared with other groups was significant(P <0.05).1.1.4 Masson staining of thrombus in inferior vena cava Collagen fibers and mucous can be dyed green, wrapped slurry, muscle, cellulose can be dyed red, the nucleolus can be dyed dark blue in Masson staining. collagen expression in thrombus was rare in 1-3 days groups. With time expanding, collagen expression area spread from edge to interior of thrombus gradually, and numerous collagen expression could be found in the whole lumen in 4weeks group. The difference was not significant in group of 3 days, 1 week and 2 weeks when compared with each other. The difference was significant in 3 weeks and 4 weeks group compared with other groups(P <0.05).1.2 Immunohistochemical staining of thrombus in inferior vena cava1.2.1 Immunohistochemical staining of CD61 CD61 expression scattered in thrombus in 3 hours group and located in platelet small beam closing to vascular wall in 6 hours group until reached the peak in 1 day group. CD61 expression decreased in 3 days and 1 week groups and was only in the position of rethrombosis in vascular lumen with second stenosis. The difference between 1 day group and other groups was significant(P <0.05)1.2.2 Immunohistochemical staining of ?-SMA ?-SMA is smooth muscle actin expressed in fibroblast during the organization of thrombus. Some ?-SMA positive expression was found in edge of thrombus in 3 days group. The expression reached peak in 1 week group. ?-SMA expressed only in some new capillary walls. The difference between 1 week group and other groups was significant(P <0.05).1.2.3 Immunhistochemical staining of CD34 CD34 is the a specific marker of vascular endothelium. CD34 expression scattered in thrombus closing to vascular wall in 3 days group firstly and accounted for 7% area of vascular lumen in 1 week group with increasing formation of new capillaries. The area of CD34 expression accounted for 22.8% of vascular lumen with red blood appearing in 4 weeks group with fusional vessels in 2-4 weeks groups. The difference between 3 days group and 1 week group was not significant. The difference of 4 weeks group compared with 3 days group and 1 week group was significant(P<0.05). But there was no difference between the 2,3 and 4 weeks groups.2 Human tissue experiments2.1 HE staining of thrombus in deep vein. Platelet aggregated in the vessel wall to form the lateral thrombus which formed the head of the mixed thrombus in the early stage of thrombosis. Then a large number of platelet aggregated to form platelet small beam. Red and white blood cells and cellulose were mixed with platelet small beam to form thrombus. With the crescent mixed thrombus blocking the lumen of vessel, the red blood cells were aggregated to form the tail of thrombus. Endothelial cells and fibroblasts grew to the interior of thrombus in the late stage. The thrombus organized. The bigger thrombus contracted and formed crannies in the interior. The endothelial cells attached to the surface of the crannies to form new capillaries and blood vessels with bigger lumen to lead to repatency of vessels. Detachment of thrombus could result in pulmonary embolism.2.2 Immunohistochemical staining of thrombus in vein vessel CD61 performed flaky expression in the pulmonary artery(a dead case in the 23 th day after trauma and fractures); positive expression of ?-SMA was observed in the edge of thrombosis position of popliteal vein(a dead case in the 31 th day after fracture); large expression of CD34 in the interior of thrombus in popliteal vein(a dead case in the 43 th day after fracture).Conclusions:1 Characteristics of thrombosis in different time were observed in both special staining(Perl's staining,Masson staining,Von Kossa staining) and immunohistochemical staining(CD61, ?-SMA,CD34) in the rats models. CD61 detection can be used in the early stage of thrombosis and thrombus reformation; Perl's staining can be used in the intermediate stage of thrombosis; Masson staining and ?-SMA detection can be used in the mid and late stage of thrombosis; Von Kossa staining and CD34 detection can be used in the late stage of thrombosis to estimate time of thrombosis. The characteristics of thrombosis provide theoretical basis for time estimation of thrombosis in forensic cases.2 The results of parts of experiments of tissue specimens from human body indicate that the above indicators and methods are meaningful for elementary study of time estimation of thrombosis in fatal cases resulted from human pulmonary embolism.