Laboratory investigation of deaths due to anaphylaxis.

To establish a useful laboratory protocol to investigate possible cases of fatal anaphylaxis, we measured mast-cell-derived tryptase levels and allergen-specific immunoglobulin E (IgE) antibody levels in sera obtained prior to or within 24 h after death from 19 anaphylaxis victims. Elevated serum tryptase levels (range = 12 ng/mL to 150 micrograms/mL) were found in nine of nine Hymenoptera sting fatalities, six of eight food-induced fatalities, and two of two reactions to diagnostic therapeutic agents. Tryptase levels were normal (less than 10 ng/mL) in 57 sequential sera obtained postmortem from six control patients. Tryptase could not be measured in pleural or pericardial fluids for technical reasons. Serum IgE antibodies were elevated in five of the nine Hymenoptera sting fatalities and in eight of the eight fatal food reactions; assays were unavailable for the two diagnostic/therapeutic agents. If elevated, the victim's serum IgE antibodies to food could be used to identify allergens in uneaten portions of foods consumed shortly before the anaphylactic event. IgE antibodies were moderately stable during storage in a variety of anticoagulants at room temperature for up to 11 weeks. Elevated mast-cell-derived tryptase levels in postmortem sera reflect antemortem mast cell activation and may be used as a marker for fatal anaphylaxis. If assays are available for IgE antibodies to relevant allergens, such assays provide evidence for antemortem sensitization; these assays may be modified to identify allergens in foods consumed by victims of food-induced anaphylaxis.     

Differential accumulation of pulmonary and cardiac mast cell-subsets and eosinophils between fatal anaphylaxis and asthma death: a postmortem comparative study

The distribution profile of infiltrated mast cell-subpopulations and eosinophils in the lung and heart sections of the patients who died of severe allergic hyperresponsiveness, was investigated. Four study groups were designed comprising 9 cases who died in systemic anaphylaxis (Group I), 10 asthmatic individuals whose death were assigned to acute and severe bronchial asthma (Group II), 10 asthmatic cases who died from non-immunological diseases (Group III). Twenty consecutive autopsies of non-allergic subjects who died of unnatural causes (Group IV) served as control group in this study. Utilizing antibodies against human tryptase and chymase and a double immunohistochemical staining method, we distinguished successfully all three subsets of mast cells (MC), MC-TC (containing both tryptase and chymase), MC-T (containing only tryptase) and MC-C (containing only chymase) types, subdivided on the basis of the protease compositions of their secretory granules. In order to immunostaining eosinophils, we used antibody to major basic protein as a marker. We also measured postmortem blood tryptase, specific and total serum IgE. The intriguing finding of this study was the marked differences of cellular composition in the lung between fatal anaphylaxis and asthma death. Significant augmentation of MCs infiltrated in lung and heart sections of anaphylaxis patients and drastic infiltration of bronchial eosinophils in asthmatic death and consequent release of their related inflammatory mediators might explain the differential expression of the associated symptoms in these two groups. The anaphylactic deaths did show neither emphysema nor significant mucous bronchial secretions whereas all asthmatic deaths did. The degree of pulmonary congestion and edema was also more severe in anaphylaxis. This corresponded with the histological findings and the location and number of mast cell-subsets and eosinophils in the different compartments of the lungs. We have demonstrated that the third type of mast cell MC-C is only found in the lungs in anaphylactic deaths. The practical consequence of our study will be that it is now possible to confirm a suspicion of anaphylaxis death not only by measurements of serum mast cell tryptase, but also by immunohistochemical methods.     

Three cases of suspected hyperthermia with remarkable elevation of serum mast cell tryptase

Tryptase is a neutral protease of human mast cells, and an important indicator of mast cell activation and degranulation in anaphylactic events. The elevation of serum mast cell tryptase (SMCT) is used for postmortem diagnosis of anaphylaxis. We have quantified the SMCT levels of 122 forensic autopsy cases with various causes of death and found only three where the SMCT levels were remarkably elevated, with values of 179, 68.9 and 69.4 ng/ml (normal level <13.5 ng/ml). The three cases were suspected to have suffered from hyperthermia, and the deaths did not seem to be related to causes of death where SMCT levels have been reported to be elevated in some cases. Two cases were patients who had been prescribed long-term neuroleptics or antidepressants, and myoglobin was detected immunohistochemically in the renal tubules of both cases. The other case died of heatstroke. A possible mechanism of hyperthermia in SMCT elevation is discussed.     

Serum tryptase levels in sudden infant death syndrome in forensic autopsy cases

An elevated serum tryptase concentration is considered to be a specific marker for systemic mast-cell activation, a central feature of anaphylaxis, which has been observed in some cases of sudden infant death syndrome (SIDS). However, it is still unclear whether anaphylaxis is involved in the etiology for SIDS. In the present study, we measured serum tryptase levels in 21 infants with SIDS, and 14 control infants from forensic autopsy cases by Uni-CAP TRYPTASE Fluoroenzyme immunoassay system, which detects both alpha- and beta-tryptase. The assay did not show any significant elevation of tryptase levels in the SIDS group compared with controls. Additionally, increased concentrations of tryptase were not observed in any SIDS case. Our results indicated that anaphylaxis does not seem to be involved in the etiology of SIDS.     

Sudden death associated with food and exercise

Exercised-induced anaphylaxis occurs in conjunction with significant physical exertion. Anaphylaxis occurring when an individual exercise within a few hours of ingesting a particular food is an unusual variant. Cardiovascular symptoms can be the sole manifestation of exercise-induced food allergies, in which case death may mimic sudden cardiac death during physical exertion due to other pathologic causes. We report the sudden and unexpected death of an individual following the ingestion of hazelnuts and almonds, to which the individual was not previously known to be allergic. The decedent collapsed during vigorous dancing. The death was not associated with cutaneous or laryngeal manifestations of anaphylaxis. Awareness of the variable manifestations of food-precipitated anaphylaxis is necessary to correctly establish the diagnosis. An elevated serum tryptase level may be indicative of an allergic reaction, and allergen-specific IgE levels may be used to confirm the particular antigen.     

Stable isotopic characterization of ammonium metavanadate (NH4VO3)

The diagnosis of fatal anaphylaxis can be difficult for clinical features may not always be evident in necropsy. Therefore post mortem determination of tryptase and other blood parameters can be helpful in verifying the diagnosis. We compared post mortem tryptase, histamine and diamine oxidase (DAO) serum levels of two patients who had died after a Hymenoptera sting and one patient who died of bronchospasm during anaesthesia with data obtained from 55 control subjects who had died from other causes than anaphylaxis. In the three anaphylactic cases, serum tryptase level was 880, 68 and 200 μg/l (normal range in living subjects: <11.4 μg/l), histamine was 37.5, 8.5 and 23.2 ng/ml (normal range: <0.3 ng/ml) and DAO was 1, 30 and 4 U/ml (normal range 10-30 U/ml), respectively. Values in the control group were as follows: tryptase 1-340 μg/l (mean 24.2 ± 58.2), histamine 5.0-22.0 ng/ml (mean 14.7 ± 3.9) and DAO 0-114 U/ml (mean 21.1 ± 27.8). 19/55 (34.5%) of the controls had elevated tryptase levels >11.4 μg/l, with four of them showing values >45 μg/ml. Significantly higher histamine levels were seen in blood samples taken more than 24h post mortem (p<0.05), whereas the timing of blood collection had no effect on tryptase and DAO levels. While moderately elevated tryptase levels are common in post mortem sera, values above 45 μg/l may support the diagnosis of fatal anaphylaxis. Strongly elevated histamine levels might give an additional clue on fatal anaphylaxis, whereas DAO does not seem to be helpful.     

类胰蛋白酶和IgE测定在过敏性猝死诊断中的应用研究

目的探讨过敏性猝死法医学鉴定的诊断方法和指标。方法采取10例正常人、9例过敏性猝死和19例其他死因（排除过敏反应、冠心病）尸体的静脉血，采用荧光酶联免疫法（Pharmacia UniCAP100过敏原定量分析仪）和酶联免疫吸附试验ELISA法分别测定血清肥大细胞类胰蛋白酶和]gE含量，采用免疫组化方法观察过敏性猝死和其他死因的肺组织中的肥大细胞类胰蛋白酶免疫组化染色。结果过敏性猝死者的血清类胰蛋白酶和IgE含量升高，与其他死因之间的差异具有显著性意义（P〈0．01），其他死因和正常人之间的差异无统计学意义（P〉0．05）；与其它死因相比，过敏性猝死肺组织中的肥大细胞类胰蛋白酶免疫组化阳性染色增强（P〈0．01）。结论过敏性猝死者血清IgE和肥大细胞类胰蛋白酶含量显著升高；过敏性猝死者肺组织中肥大细胞类胰蛋白酶染色增强。     

An Analysis of Possible Mechanisms of Unexpected Death Occurring in Hydatid Disease (Echinococcosis)

Abstract:  Most cases of hydatid disease in human populations are due to Echinococcus granulosus. The hydatid life cycle involves passage between definitive hosts such as dogs and intermediate hosts such as sheep. Humans become accidental intermediate hosts following ingestion of food or water contaminated with eggs or by contact with infected dogs. Although hydatid disease may remain asymptomatic, occasional cases of sudden and unexpected death present to autopsy. Causes of rapid clinical decline involve a wide range of mechanisms including anaphylaxis (with or without cyst rupture), cardiac outflow obstruction or conduction tract disturbance, pulmonary and cerebral embolism, pericarditis, cardiac tamponade, myocardial ischemia, pulmonary hypertension, peritonitis, hollow organ perforation, intracerebral mass effect, obstructive hydrocephalus, seizures, cerebral ischemia/infarction, and pregnancy complications. The autopsy assessment of cases therefore requires careful examination of all organ systems for characteristic cystic lesions, as multiorgan involvement is common, with integration of findings so that possible mechanisms of death can be determined. Measurement of serum tryptase and specific IgE levels should be undertaken for possible anaphylaxis.     

免疫正常人和过敏性猝死者类胰蛋白酶含量测定

目的观察免疫正常人和过敏性猝死者血清类胰蛋白酶、IgE含量及类胰蛋白酶在咽喉、肺和小肠组织的表达,探讨过敏性猝死的诊断方法。方法采用荧光酶联免疫法（FEIA）测定115例正常人和6例过敏性猝死者血清类胰蛋白酶;采用酶联免疫吸附试验（ELISA）测定7例过敏性猝死者血清IgE含量;采用免疫组织化学方法观察15例过敏性猝死者咽喉、肺、小肠组织中类胰蛋白酶的表达,并采用BL-2000病理图像分析系统进行定量分析。结果①115例免疫正常人血清中类胰蛋白酶含量在0.80μg/L～8.86μg/L之间,性别之间无明显差异（P〉0.05）。20岁以下、21～40岁和41～60岁组之间血清中类胰蛋白酶含量无明显差别（P〉0.05）,而60岁以上组含量高于其他年龄组（P〈0.05）;②6例过敏性猝死者血清类胰蛋白酶含量均高于免疫正常人;③7例过敏性猝死者中有6例血清IgE水平高于正常值（〉333U/mL）;④过敏性猝死者咽喉、肺和小肠组织中类胰蛋白酶表达升高,与对照组之间有显著性差异（P〈0.01）。结论过敏性猝死者血清IgE、类胰蛋白酶含量及咽喉、肺和小肠组织类胰蛋白酶表达升高,可对敏性猝死的诊断有一定参考价值。     

不同温度下大鼠脑组织RNA降解与早期PMI的相关性

目的探讨大鼠脑组织8种RNA指标,在不同温度下的表达水平与早期死亡时间(PMI)的相关性。方法将222只SD大鼠随机分为对照组(死后0 h)和4个实验组,实验组断颈处死后分别置于5℃、15℃、25℃和35℃的环境中,于死后1~24 h内9个时间点提取脑组织RNA,利用实时荧光定量PCR技术检测8种RNA指标(β-actin、GAPDH、RPS29、18S rRNA、5S rRNA、U6 snRNA、miRNA-9及mi RNA-125b)的表达水平,ge Norm软件选取合适内参,SPSS软件对内参标准化RNA指标进行回归分析,R软件构建推断PMI的数学模型,另选6只已知PMI的SD大鼠予以验证。结果 5S rRNA、miR-9和mi R-125b表达稳定,可作为内参指标。β-actin和GAPDH具有良好的时序性降解规律,在24 h内随PMI延长不断降解。R软件拟合得ΔCt值随PMI和温度变化的数学模型可用以推断PMI。运用β-actin和GAPDH验证模型的平均误差率分别为14.1%和22.2%。结论β-actin和GAPDH表达水平与PMI和环境温度相关性良好。本研究建立的数学模型可为温度变化条件下的早期PMI推断提供参考。     

组织RNA检测用于死亡时间推断的研究进展

近几年来利用RNA的降解程度来推断死亡时间已逐渐成为法医学研究热点.本文复习相关研究文献,对大鼠不同组织(脑组织、心脏组织、肝脏组织和肾组织等)中RNA在推断死亡时间的具体应用方法做出总结,归纳出不同组织中的各类RNA在推断死亡时间方面的适用范围,以及对此种方法在推断死亡时间的过程中产生的问题及可行性的解决方法.     

Vitreous humor biochemical constituents: evaluation of between-eye differences

The present study was conducted to investigate the differences in the vitreous humor biochemical concentrations for vitreous electrolytes and calcium in the same pair of eyes at identical postmortem interval (PMI). The vitreous humor samples were collected independently in both eyes from 48 autopsies (PMI range, 4.5-84.3 hours) with documented time of death. The samples were analyzed for potassium, sodium, chloride, and calcium using a Beckman Coulter LX20 Automated Analyzer based on ion-selective electrode methodology. There were no statistically significant between-eye differences at identical postmortem interval. A significantly high correlation was observed between paired potassium concentrations of both the eyes. A highly significant linear correlation was observed between the individual eye and mean potassium concentrations of both the eyes with postmortem interval. The observed differences were not significantly correlated with postmortem interval. The results demonstrated that the between-eye differences for vitreous electrolytes and calcium are insignificant. Therefore, the utility of vitreous biochemistry, particularly potassium in postmortem interval estimation and other forensic applications, cannot be questioned solely on the basis of these differences.     

大鼠死后管家基因mRNA稳定性及其时序性降解与PMI相关性研究

目的研究脑组织及骨骼肌内β-actin、GAPDH、RPL32、PKG1、SDHA、RPL13、HPRT、TBP、YWHAZ死后稳定性及其相对表达量与死亡时间(PMI)的相关性。方法取健康成年SD大鼠33只,依据据死亡时间不同随机分为11组(0h、1h、3h、6h、12h、24h、2d、4d、8d、16d、24d),分别在各时间点取大鼠胫骨前肌及脑组织约50mg,提取各组织总RNA,利用RT-q PCR技术检测9种管家基因的m RNA相对表达量。运用ge Norm、Norm Finder和Best Keeper软件对基因稳定性进行评价,利用spss软件对内参标准化的m RNA指标进行回归分析。结果脑组织及骨骼肌中均为HPRT稳定性最高,适合用于本研究的内参基因。脑组织中SDHA、RPL32及TBP 3种指标的相对表达量变化与PMI有一定的线性关系。骨骼肌中m RNA指标相对表达量与PMI之间没有显著的相关性。结论脑组织及骨骼肌是较适宜推断晚期死亡时间的检材。脑组织中SDHA、RPL32及TBP 3种m RNA的相对表达量变化与PMI有一定的线性关系,有望成为推断PMI的辅助指标。     

大鼠死后脑、心肌和肾组织β-actin mRNA的降解与早期死亡时间的关系

目的 研究SD大鼠脑、心肌和肾组织细胞内β-actin mRNA的降解与早期死亡时间的关系,为早期死亡时间的推断寻找新的指标.方法 大鼠处死后置于20℃的环境中,分别于死后不同时间点提取脑、心肌、肾的总RNA,采用实时荧光定量RT-PCR方法检测总RNA中β-actin mRNA的水平(Ct值),分析死后经过时间与Ct...     

Mast cell tryptase and hemolysis after trauma

BACKGROUND: We have previously found increased mast cell tryptase in accidental deaths due to trauma, indicating that mast cell degranulation had occurred. The present study was designed to confirm the previous observation and to determine if tryptase release after trauma is acute or delayed. Furthermore, the importance of hemolysis and direct trauma to the mast cells was investigated. MATERIALS AND METHODS: Mast cell tryptase was measured in post-mortem blood from the femoral vein in 27 cases of death from trauma and in 27 control cases by means of a commercially available immunoassay. The trauma cases were further classified into groups with single versus multiple trauma, and groups with short survival time (i.e. death at the scene of the accident) versus longer survival time (death in hospital). In five multi-trauma deaths, blood was sampled locally from the sites of crush injury. RESULTS: The mean value of tryptase in femoral vein blood was 35.6+/-34.6 microg/l in the entire trauma group and 14.7+/-6.5 microg/l in the controls (P<0.005). In bloody liquid sampled from crush injuries, tryptase was substantially elevated in all cases, with a mean of 227+/-146 microg/l. In cases with short survival time, tryptase was significantly higher than in those who died after several hours or days in hospital (P<0.001). No statistically significant difference was seen between multi- and single-trauma cases. A correlation between hemolysis in the samples and elevated tryptase was found only in the trauma cases (P<0.05), but experimentally induced hemolysis in vitro was not found to influence the measurements. CONCLUSION: Mast cell tryptase becomes elevated in trauma deaths and this seems to be ascribable either to direct mechanical injury to tissue mast cells and/or to cell lysis. In patients initially surviving severe injuries, the effects of massive release of histamine and other mast cell mediators might be of importance for treatment strategies and prognosis.     

An Examination of the Transition of Fracture Characteristics in Long Bones from Fresh to Dry in Central Florida: Evaluating the Timing of Injury

It is important to conduct timing of injury research analyzing fracture characteristics at known postmortem intervals (PMI) because bone can retain fresh characteristics throughout the PMI. Defleshed pig (Sus scrofa) long bones were fractured weekly in two environments (full sun and shade) over 14 weeks in Central Florida and fracture characteristics were categorized (N = 136) for analysis. Results of analysis of variance (ANOVA) using time in weeks (PMI) as a dependent variable indicate significant relationships between PMI and Fracture Angle (p < 0.001), Fracture Surface (p < 0.001), and Fracture Outline (p < 0.001). Fracture characteristics associated with perimortem trauma (smooth Fracture Surfaces and curved or V‐shaped Fracture Outlines) were commonly observed. Analysis of fracture characteristics for each environment demonstrated similar patterns. Overall, the loss of only fresh fracture characteristics for each bone was noted earlier in the PMI for the Central Florida region than previously reported.     

The changes of fourier transform infrared spectrum in rat brain

Estimation of the time since death (postmortem interval [PMI]) is one of the most difficult problems in forensic investigations, and many methods currently are utilized to estimate the PMI. The goal of this study was to investigate the changes of attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of rat brain from postmortem time 0-144 h. The intensity ratios of major absorbance bands were examined (I(1066)/I(1392), I(1168)/I(1392), I(1234)/I(1454), I(1301)/I(1392), I(1647)/I(2956), I(2921)/I(2850), and I(1647)/I(1539)). The spectra of rat brain displayed prominent changes with increasing PMI. The band at 2871 and 1737 per cm became weak with the time increasing and even disappeared at postmortem 96 and 72 h, respectively. A close linear correlation was shown between the relative absorption intensity and the PMI, and the I(1234) /I(1454) offered a stronger correlation (r = 0.973). Our results indicate that ATR-FTIR spectroscopy may be a useful technique for estimating the PMI.     

Estimating the timing of long bone fractures: correlation between the postmortem interval, bone moisture content, and blunt force trauma fracture characteristics*

There is very limited knowledge about how long perimortem fracture characteristics persist into the postmortem interval (PMI). Therefore, in this study, 60 porcine long bones were exposed to natural taphonomic conditions and fractured with a steel bone breaking apparatus every 28 days throughout a 141-day period. Differences between macroscopic blunt force trauma fracture characteristics (fracture angle, surface morphology, and outline) were examined to determine if they varied over time or in relationship to bone moisture content (ash weight) and overall assessment. There are significant relationships between (1) PMI and percent ash weight (%AW), fracture surface, and fracture angle and (2) %AW and fracture surface and fracture angle. Bone moisture content correlates significantly with fracture morphology and other characteristics commonly used by forensic anthropologists to determine the timing of traumatic injuries. However, fracture characteristics normally associated with perimortem trauma can persist long into the PMI.     

Postmortem Serum Tryptase Levels with Special Regard to Acute Cardiac Deaths

An elevated serum tryptase concentration is considered a specific marker for systemic mast cell activation, a central feature of anaphylaxis. However, in some cases of acute cardiovascular death, high concentrations of serum tryptase are also observed. We compared the postmortem serum tryptase concentrations in 74 cases assigned to the following four groups: anaphylactic deaths (Group A, n = 20), acute cardiac deaths (Group ACD, n = 30), acute dissecting aneurysm ruptures (Group ADA, n = 10), and controls (Group C, n = 14). Additionally, the cutoff between Group A and the other groups was calculated using receiver‐operating characteristic (ROC) curve analysis. Tryptase concentrations were markedly elevated in Group A (p < 0.001), Group ACD (p = 0.015), and Group ADA (p = 0.005). The optimal cutoff was 43 ng/mL, the sensitivity was 90%, and the specificity was 98%. While elevated concentrations of tryptase were noted in practical autopsy cases, due attention should be paid to the differential diagnosis between anaphylactic and acute cardiovascular deaths.     

Sudden infant death syndrome: measurement of total and specific serum immunoglobulin E (IgE)

Postmortem evaluation of total and specific serum immunoglobulin E (IgE) antibody levels by the paper radio immuno sorbent test (PRIST) and radio allergo sorbent test (RAST), respectively, revealed that there was no significant elevations in total circulating IgE or in specific IgE antibodies to house dust, Dermatophagoides farinae (house dust mite), Alterarnia tenuis (mold), or milk proteins for sudden infant death syndrome (SIDS) victims when compared to a control group.