| BACKGROUNDAnaphylaxis is severe, life-threatening, generalized or systemic and immunologically mediated hypersensitivity reaction that occurs suddenly after exposure to certain foreign substances in previously sensitized persons. The most common life-threatening feature of acute anaphylaxis is cardiovascular collapse or shock, the other life-threatening effects include laryngeal oedema, bronchospasm, angio-oedema and pulmonary oedema. Immediate hypersensitivity is a clinical emergency which is sudden and life-threatening. Data on anaphylaxis incidence and prevalence of American:the frequency of anaphylaxis is approximately50to2,000episodes per100,000persons (excluding the undocumented, undetermined and misdiagnosed patients), and there are1,500anaphylactic death every year. Epidemiology of anaphylaxis in our country is not enough detail. In many forensic cases of fatal anaphylaxis no specific macroscopic findings are present at postmortem examination. Unlike clinical purposes, a post-mortem diagnosis of anaphylaxis turns out to be very difficult, medico-legal investigations on a suspected anaphylactic death should be essentially focused on a perusal of the scene, history of exposure to allergens and the clinical history, followed by the evaluation of necroscopic and laboratory data, and after the objective exclusion of other possible fatal causes different from anaphylaxis.There were lots of researches about the relationship between the mast cells granule-associated mediators (serum total IgE, histamine, tryptase, chymase, leukotrienes, thromboxane B2, substance P) and anaphylactic reaction, the stability of those mediators in vitro have been reported, but the concentration and stability of those mediators may be modified under the effects of different thanatological processes, which may cause cytolysis and chemical degradation during the post-mortem period.OBJECTIVEMost of the forensic autopsies are carried on few days after death, how do the related-indicators of anaphylaxis change after death? There have been reported that the serum mast cell tryptase becomes elevated in non-anaphylactic deaths with significant atherosclerosis or severe trauma with increasing PMI. Some research show that the IgE in serum and lung after guinea-pig anaphylactic death did not change within48h in freezing conditions. The studies on the stability of anaphylactic related-indicators focus on serum in vitro at room temperature or serum in vivo in freezing conditions, few studies show the change of IgE, tryptase and chymase in serum or tissue at room temperature with the increasing PMI.According to the literature, we established mixed human serum-induced fatal anaphylactic reaction model in guinea-pig and made appropriate improvement. To confirm the model being successfully established, we observed the anaphylactic reaction of the guinea-pigs, pathological change of tissue and detected the levels of serum IgE and tryptase using the ELISA. Based on the improved model, we used the ELISA to measure the concentration of serum IgE, tryptase and chymase, finally discussed the changes of IgE, tryptase and chymase in guinea pig died of anaphylaxis at room temperature within24h. METHODS1. Establisment and the improvement of the animal model and observation of the anaphylactic reactionSeventy four Guinea pigs, weighting from250g to300g (the experimental animal centre of Southern Medical University, China) were randomly divided into the original model groups (twenty five experimental groups and eight negative control groups) and the improved model groups (twenty five experimental groups with eight negative controls and eight non-anaphylaxis controls). The guinea pigs were fed a week before experiment. The original experimental groups were intracutaneously injected with allergen (0.15ml, the proportion of mixed serum and NS is1:10), after feeding three weeks at room temperature, the same allergen(lml) was intracardiac injected to induce the anaphylaxis. The original control groups were the same administration as experimental groups, but with saline instead of the allergen. The improved experimental groups were intracutaneously injected with allergen (0.1ml, the proportion of mixed serum and NS is1:2) every hind claw palm, the negative control groups and the non-anaphylaxis control groups were the same administration with saline instead of the allergen. One day after the injection, the same treatment was carried on again. Two weeks after the second injection, lml allergen (1:20) was intravenously injected (dorsal vein of foot) in original experimental groups and non-anaphylaxis control groups, and the original negative control groups were the same treatment with saline instead of the allergen.Record the anaphylactic reaction of guinea pigs. Macroscopic and histological investigations were taken after the death. Blood was taken to determine the levels of serum IgE, tryptase and chymase by ELISA.2. Use ELISA to test the level of IgE, tryptase and chymase in serum which was respectively in the guinea pigs’corpses, incubating at room temperature in vitro and in the lung and gastric wall.(1) Test the level of serum IgE, tryptase and chymase in the guinea pigs’corpses. Based on the improved model,114guinea-pigs were classified into six time stages: Oh,2h,4h,8h,12h and24h, each stage randomly divided into11experimental guinea-pigs and8control groups. All the guinea pigs’carcasses were placed onto a custom-made cases with wire mesh at room temperature and dissected at different postmortem interval respectively. Blood was taken to determine the levels of serum IgE, tryptase and chymase within24h by ELISA.(2) Determine the level of IgE, tryptase and chymase in serum incubated at room temperature in vitro by ELISA. Blood of0h group was stood and centrifuged to take the supernatant. Both of the supernatant was cut into four equal parts. One of the serum’s level of IgE, tryptase and chymase was tested using ELISA, and the others were tested after placing at room temperature for1d,2d and3d.(3) Level of IgE, tryptase and chymase in guinea pigs’lung and gastric wall tissue. Lung and gastric wall tissue was removed from six experimental and control goups’body,100mg tissue were washed by normal saline and stored at-80℃after freezing-and-thawing three times. Before ELISA, sample were thawed and homogenized, centrifuged(15000rpm for30min) at4℃, and the supernatant removed to test the level of IgE, tryptase and chymase.RESULT1. After the inducing anaphylaxis,18improved model guinea pigs and13original model groups started to appear severe anaphylactic reaction (at first remained quiet, but within one minute restlessness developed, giving frequently a spasmodic moist ringing sneeze, it sat up on its hind legs and vigorously rubbed the nose and head, violent tonic and clonic convulsions developed, occasionally there was a jump, and the animal can no longer stand, but fell on its side the legs. The mucous membranes of the mouth and the tongue become bluish, oral and nasal secretions increased, and the mouth opened with each respiration, which was very slow and finally ceased. The heart still beat regularly for many minutes after all respiration have stopped.). But the cardiac arrest of the improved groups was4minutes later than the original groups. Mild symptoms were happened in two guinea pigs of the improved groups and one of the original groups, all of them recovered20minutes later. We found that the incidence of anaphylactic death in improved groups was75%,and the original groups was54.2%. With the necroscopic investigation, laryngeal edema, severe emphysema and congestion was found in most of the guinea pigs died of anaphylaxis. Eosinophilia was present at larynx, trachea, bronchi and gastrointestinal submucosa. All guinea-pigs of original groups appeared hemopericardium and five guinea-pigs died of severe pericardial tamponade. There was no significantly different in serum IgE between the original experimental groups and negative control groups. Compared with the original experimental groups, levels of serum IgE increased significantly in improved experimental groups (F=16.68, P<0.001). Tryptase in improved experimental groups was higher than that of original experimental groups (F=18.268, P<0.001).2.(1) After incubation in vitro at room temperature, No statistically significant difference was seen in serum IgE(F=0.386, P=0.764>0.05), tryptase(F=0.005, P=0.999>0.05) and chymase(F=0.145, P=0.932>0.05) within three days.(2) Statistically significant difference of serum total IgE, tryptase and chymase level were existed between the control groups and the test groups (P<0.001). No statistically significant difference was seen in test groups’ serum total IgE and chymase at different postmortem interval within24h (P>0.05). There was statistically significant difference in test groups’ serum tryptase in24h (P<0.05).(3) No statistically significant difference was seen in lung IgE(F=0.173, P=0.686>0.05) between the test and control groups. Compared with the control groups, the level of tryptase (F=31.92, P<0.05) and chymase(F=6.989, P=0.025<0.05) elevated significantly. There is no statistically significant difference of IgE (F=1.537, P=0.243>0.05) and tryptase(F=0.989, P=0.334>0.05) in gastric wall tissue between the test and control groups. Chymase of gastric wall tissue in test groups was much higher than the controls(F=65.858, P<0.05).CONCLUSION1. The mortality of anaphylaxis had been improved through changing the measures of sensitization. There was no hemopericardium or tamponade because the allergen was injected through peripheral veins. The improved level of the IgE and tryptase makes the animal model of anaphylaxis more efficiency.2.(1) According to the statistical result, there was no remarkable change of the level of IgE, tryptase and chymase in serum incubated at room temperature within three days. The level of IgE, tryptase and chymase had been shown to be relatively stable in vitro within three days.(2) No statistically significant difference was seen in serum total IgE within24h in vivo at room temperature. That may contribute to the production and elimination of the IgE in vivo after death. IgE is stable at room temperature within three days. It may ascend after challenged with allergens, but it would not be generated as soon as the anaphylaxis. And the main way of elimination of IgE is the intra-vascular and extra-vascular redistribution. So the stable characteristic and slow elimination after death may contribute to IgE with no significant difference within24h.Tryptase is a neutral protease which is characteristic of mast cells’activation. The result of our study showed that tryptase have a rising trend within24h after death in vivo at room temperature(F=2.881, P=0.018<0.05). We know that the lung and heart is the target organ of anaphylaxis in many species because of much mast cell locating in pulmonary and cardiac tissue. Mast-cell related mediator excretes into intercellular matrix from sensitized mast cell as soon as anaphylaxis. As the coming of anaphylaxis, tryptase from active mast cell of pulmonary and cardiac tissue can release into intercellular matrix, and the mediator may diffuse from matrix into vessel after death.Chymase is a serine protease which also present in the secretory granules of mast cells. Statistically significant difference of chymase level were existed between the control groups and the test groups serum(F=127.608, P<0.001) and lung(F=6.989, P=0.025<0.05). The main mast cell in lung is MCT which only secrete tryptase. That the subunit of mast cell in lung and host defense function of the chymase may both contribute to the result. There is no significant difference of serum chymase within24h after death in vivo at room temperature, maybe contribute to that the subunit of mast cell in lung and few chymase can diffuse after death. |
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