勒福特王水消解法检验动物器官硅藻的法医学应用

目的研究勒福特王水消解法对动物器官中硅藻检验的法医学应用。方法将勒福特王水消解-滤膜富集-扫描电镜观察法与传统强酸消解-离心富集-光学显微镜观察法比较,对生前入水和死后入水的实验动物肺、肝和肾组织中的硅藻数量、检验时间、检出率以及观察效果进行研究,并进行统计学分析。结果生前入水组用勒福特王水消解-滤膜富集-扫描电镜观察法检出肺、肝、肾组织中的硅藻数量均多于传统方法(P0.05),检验时间少于传统方法(P0.05),检出率无差异(P0.05),观察效果优于传统方法。结论勒福特王水消解-滤膜富集-扫描电镜观察法具有简单、高效、定性定量分析准确的特点,该方法成本低廉,操作简单,具有较好的实用价值。     

强酸消化法和胰蛋白酶消化法检验腐败脏器硅藻的比较研究

目的探讨强酸消化法和胰蛋白酶消化法检验腐败脏器硅藻的优缺点，为法医工作者选择硅藻检验方案提供理论依据。方法用24只健康清洁级家兔，雌雄不限，制作溺死动物模型。室温放置72h后，分别取其肝、肾、肺3种组织各5g，用强酸消化法和胰蛋白酶消化法进行消化，分别比较两种消化方法的消化时间、消化能力和硅藻检出率。结果强酸消化法的消化时间显著短于胰蛋白酶消化法（P〈0．05），强酸消化的消化能力强于胰蛋白酶消化法（P〈0．05），而强酸消化法的硅藻检出率低于胰蛋白酶消化法（P〈0．05）。结论为加快办案效率，可以选择强酸消化法；对于水样硅藻含量低，则选择硅藻检出率相对较高的胰蛋白酶消化法。     

Detection of Diatoms by a Combination of Membrane Filtration and Transparentness,

Diagnosis of drowning remains a difficult issue in current forensic sciences. A large number of diatoms were lost by removing the supernatant after centrifugation in the conventional forensic diatom test. We developed a novel membrane filtration method to enrich diatoms from samples. A new solution using different ratios of acetic acid and eugenol is prepared to make the membrane transparent. These processes allow the diatom‐containing membrane to be visualized and identified easily by light microscopy. The tissues contaminated by water rich in diatoms were detected by the new method for the recovery of diatoms. Eleven drowning cases were analyzed by both the new method and the conventional method to compare the sensitivity of both methods. The recovery of the novel diatom test method was 54.2 ± 23.1%. The positive rate of the novel method has been proven to be superior to the conventional method in the diagnosis of drowning.     

4种硅藻检验方法的比较研究

目的比较硝酸乙醇法、酶消化法、“破机罐”法和Soluene-350法在检验硅藻中各自的优缺点,为法医实际办案提供理论依据。方法取家兔肝、肾和骨髓3种组织各2g,分别用上述4种方法消化,比较4种不同方法分别将组织这些完全消化所需要的时间、消化能力、对硅藻的检出率以及对硅藻的破坏程度。结果消化时间以Solu-ene-350法耗时最长,其次是酶消化法,硝酸乙醇法和破机罐法耗时最短。4种方法消化能力的强弱依次是“破机罐”法、硝酸乙醇法、酶消化法、Soluene-350法。对梅尼小环藻(淡水)和桥弯藻(淡水),酶消化法的检出率最高,硝酸乙醇法其次,“破机罐”法和Soluene-350法最低。对舟形藻(海水),酶消化法的检出率高,其余3种方法几乎没检测到。扫描电镜观察:酶消化法对硅藻结构几乎没有破坏。其余3种方法对硅藻均有不同程度的破坏。结论从降低成本、提高检测效率角度,应选择“破机罐”法和硝酸乙醇法;从安全性和环保角度,或可疑水样为海水及硅藻密度较小时,应选择酶消化法;Soluene-350法因耗时长、成本高且硅藻检出率低,不适于在基层法医中推广。     

The Diagnostic Value of Quantitative Assessment of Diatom Test for Drowning: An Analysis of 128 Water‐related Death Cases using Microwave Digestion‐Vacuum Filtration‐Automated Scanning Electron Microscopy

The value of diatom test for the diagnosis of drowning remains controversial. The conventional forensic diatom test with low sensitivity is not a useful tool to provide accurate information about diatom in the tissues and organs. To improve the sensitivity of the diatom test, we developed a novel method called the Microwave Digestion‐Vacuum Filtration‐Automated Scanning Electron Microscopy (MD‐VF‐Auto SEM) method which resulted in a high recovery of diatoms. In this article, we analyzed 128 water‐related death cases. Our results showed that the MD‐VF‐Auto SEM method could achieve a much higher positive rate (0.97) in drowning cases. Large amounts of diatoms are retained, even concentrated, in the lung tissues during the intense breathing movement in drowning process. This might be useful for the diagnosis of drowning. Our research indicates that the MD‐VF‐Auto SEM method would be a valuable methodology in the study of diatom test for the forensic community.     

Evaluation of four digestive methods for extracting diatoms

Objective

To compare the merit of four digestive methods (nitric acid plus hydrogen peroxide, proteinase K, nitric acid in Disorganization Can and Soluene-350) for extracting diatoms in order to choose the best digestive method for the diagnosis of drowning.

Methods

Liver, kidney and bone marrow of rabbits were minced and then digested by four digestive methods separately with the following indices compared: (1) time demanded for complete digestion; (2) degree of digestion for different tissues; (3) the reclaiming ratio of diatoms; (4) the degree of digestive destruction to diatoms.

Results

For sufficiently digesting the same tissue, the demanded times for the different methods ranked from the longest to the shortest were as follows: Soluene-350, proteinase K, nitric acid plus hydrogen peroxide, nitric acid in Disorganization Can. Nitric acid in Disorganization Can method and nitric acid plus hydrogen peroxide method digested the tissues more thoroughly than proteinase K, than Soluene-350 methods. For Cyclotella and Cybella, proteinase K method reclaimed most diatoms and nitric acid plus hydrogen peroxide method reclaimed less, while nitric acid in Disorganization Can and Soluene-350 methods reclaimed the least. For Navicula, the majority of diatoms could be extracted using proteinase K method, but only a few diatoms with other three methods. Under scanning electron microscopy (SEM), the structure of diatoms remained almost perfect after digestion with proteinase K, but destroyed to some extent with other three methods.

Conclusion

This study demonstrated that different diatoms (in fresh or sea water) have different resistance to different digestive reagents. As far as the reliability and applicability of the diatom test is concerned, proteinase K method is of the best choice, nitric acid plus hydrogen peroxide can be its substitute. Soluene-350 cannot be used for extracting sea water diatoms.     

微波消解-扫描电镜联用法在溺死诊断中的应用

目的探讨微波消解-扫描电镜联用法在溺死诊断中的应用价值。方法收集已知死因的尸体标本105例,其中水中尸体85例(生前溺死70例,死后抛尸入水15例),陆地自然死亡尸体20例。水中死亡案例同时收集落水处水样。分别用微波消解-扫描电镜联用法(方法 A)和硝酸破机-光镜联用法(方法 B)对上述尸体的离体肺、肝、肾、骨髓组织及水样进行硅藻定性、定量检测。结果①溺死尸体的肺、肝、肾、骨髓中及落水处水样硅藻检出率:A法分别为100%、94.3%、92.9%、82.9%、100%,硅藻检验阳性率为100%;B法分别为90%、62.9%、51.4%、28.6%、92.9%,硅藻检验阳性率为65.7%。②两种方法检出的硅藻种类与落水处水样中硅藻的种类均一致。③两种方法在死后入水尸体离体的肺中也检出少量硅藻(<3个/2g肺组织),但在死后入水尸体的其它脏器及陆地自然死亡尸体脏器中均未检出硅藻。结论微波消解-扫描电镜联用法较硝酸破机-光镜联用法对尸体离体组织脏器中的硅藻检出率高,方法灵敏,定性准确。     

溺死法医学检验鉴定方法评估与展望

综述近年来在硅藻检验、水中浮游生物叶绿素（A）检测、血液化学和组织化学检验等方面的最新文献报道,并对各种溺死检验方法的优缺点进行了评价：在硅藻检验中,硝酸乙醇法、破机罐法及微波消解法,可缩短检验时间,提高办案效率;酶消化法及PCR法硅藻检出率高,适用于可疑水样中硅藻密度低样品的检测。早期器官组织中浮游生物叶绿素（A）、血液和组织中其他生化指标,可作为鉴定溺死的重要参考;微量元素锶检测可用于鉴定海水中溺死;另外,硅藻及其他浮游生物遗传多态性片断PCR,可望成为新的、灵敏的溺死检测方法。     

Effect of Sex Offenders Treatment Program on Cognitive and Emotional Characteristics of Mentally Ill Sex Offenders

This study assessed the effect of a 10‐week cognitive behavior treatment program in 30 mentally ill sex offenders. The effect of the program was evaluated using the Interpersonal Responsiveness Index (IRI), UCLA Loneliness Scale (UCLALS), Coping Using Sex Inventory (CUSI), and Rape Myth Acceptance Scale (RMAS). Data were analyzed using the paired t‐test. The ability of sex offenders to cope with sexual acts when they faced stressful situations and to accept the rape myth was significantly improved on CUSI (t = 2.09, p = 0.04) and RMAS (t = 5.45, p < 0.001). Feelings of isolation and the ability to empathize based on IRI (t = 0.62, p = 0.54) and UCLALS (t = 0.88, p = 0.38) were not significantly improved. To prevent recidivism, treatment for mentally ill sex offenders should focus on changes in their cognitive and emotional characteristics in addition to their main psychiatric illness.     

溺死尸体器官硅藻检出率季节性差异

目的 研究溺水死亡者肺、肝、肾、骨髓等器官淡水硅藻检出率可能存在的季节性差异,为法医学解决水中尸体死因鉴定提供技术帮助与科学证据.方法 收集2009～2011年1、4、7、10月沈阳市浑河中溺死尸体每月(组)各5例;45只家兔分相应月份生前、死后入水组及对照组,分别于当月发现水中尸体后第2天入水,取人及兔肺、肝、肾、股骨骨髓及现场水样备检,应用酶消化法检测各组器官中硅藻数量、种属并与现场水样比对.结果 各组溺死尸体肺中均检出多量硅藻且种属与现场水样一致,春、秋及冬季溺死尸体肝、肾中仅可检出少量体积较小的小环藻等中心纲硅藻,夏季肝、肾及骨髓中均未检出硅藻;各组溺死家兔肺中均检出少量小环藻等中心纲硅藻,肝、肾及骨髓中未检出硅藻.结论 硅藻种群的季节性变化及不同种群硅藻体积的差异可能造成肝、肾等大循环器官中硅藻检出率的季节性差异;仅肺中检出硅藻,肝、肾中硅藻检验结果阴性并不能排除溺死可能.     

Detection of diatom in formalin-fixed tissue by proteinase K digestion

The diatoms detection has been proposed to be useful in the diagnosis of drowning. Enzymatic digestion of unfixed lung tissues and other organs with proteinase K is widely employed to detect diatoms. Handling unfixed organs or blood from the bodies with some infectious diseases could prove to be dangerous. In this study, we examined the application of enzymatic digestion for diatom detection to formalin-fixed lung obtained at autopsy. Furthermore, we assessed the effect of hydrogen peroxide on the contamination of the lung specimen with foreign bodies inhaled in the course of drowning, smoking, or air pollution. Formalin-fixed lung was heated in 0.01 M Tris–HCl buffer (pH 7.5) containing sodium dodecyl sulfate (SDS) (tissue lysis-buffer), with or without glycine. Thereafter, the lung was subjected to enzymatic digestion with proteinase K. A part of formalin-fixed or unfixed samples digested with proteinase K were incubated with hydrogen peroxide at 80 °C for 6 h or 12 h, while the residues were processed without incubation. Formalin-fixed samples heated in tissue lysis-buffer with glycine could be digested with proteinase K; further, the number and proportion of diatoms detected in both formalin-fixed and unfixed samples were observed to be similar. The results suggest that enzymatic detection of diatoms can be applied to formalin-fixed organs by heating the samples in glycine-containing tissue lysis-buffer. As the use of formalin-fixed tissue for diatom detection can decrease risk of contamination by pathogenic organisms during the course of enzymatic digestion, the method presented in this study would be beneficial, to some extent, to individuals performing diatom analysis. Moreover, our results suggest that archival organs stored in formalin solution could be available in diatom detection over a long time-period following autopsies. Clearer image of diatoms was observed in the specimen incubated with hydrogen peroxide for 6 h, in which inhaled foreign bodies were discolored, than those not subjected to incubation. Therefore, incubation of sample digested with hydrogen peroxide in the limited time would be helpful for quantitative and qualitative diatom analysis.     

德州市水域中硅藻分布的研究

目的研究德州市水域中硅藻分布的特点,建立德州市春季和夏季的各水域硅藻数据库。方法对德州市主要水域的130个取水地点进行春季和夏季两次采样,用强酸消解-真空抽滤-扫描电镜法检测各水域中硅藻的种属及含量,统计分析水域中硅藻含量、群落组成、相对丰度和优势种属特点。结果夏季的硅藻含量高于春季的硅藻含量,河流两个季节硅藻含量无统计学显著差异(P>0.05),湖泊和水库两个季节硅藻含量存在统计学差异(P<0.05)。各水域之间硅藻含量存在差异,含量最高为608020个/10mL,最少为10个/10mL。各水域之间种属组成、优势种属和种属优势率存在差异。结论德州地区不同水域、不同季节的水中硅藻种属和含量具有明显的特异性;建立硅藻数据库和掌握本地区水域中硅藻种属特点,有助于德州市水域内水中尸体案件硅藻的检验和落水地点的判断。     

Radiodense bullet wipe around osseous entrance gunshot wounds

“Bullet wipe” is the material deposited by a bullet on any surface with which it comes into contact after it is fired and may contain debris from the gun barrel, including particles of primer and metal fragments from previously fired bullets. X‐ray analysis is a non‐destructive method by which traces of metallic elements can be visually detected. The analysis of osseous defects for radiodense bullet wipe (RBW) assists in determining the presence or absence of perforating gunshot wounds, especially in fragmented, skeletonized remains. The aim of our current study was to determine the frequency of RBW around entrance firearms injuries that perforated bone. We prospectively analyzed entrance gunshot wounds for RBW over a three‐year period using digital X‐ray analysis (n = 59). We retrospectively reviewed the corresponding autopsy reports to determine the frequency of RBW by biologic sex, reported ancestry, age‐at‐death, location of wound, manner of death, range of fire, bullet caliber, and presence of bullet jacket. Data were analyzed by Fisher's exact test or Chi‐square test with significance levels accepted at p < 0.05. RBW was present in 66% (n = 39) of examined cases. Decedent characteristics did not significantly alter RBW distribution, including biologic sex (p = 0.75), reported ancestry (p = 0.49), and age‐at‐death (p = 0.43). Additionally, the location of the osseous entrance gunshot wound, manner of death, range of fire, and cartridge caliber did not affect RBW detection. All cases involving non‐jacketed rounds (n = 5) showed RBW (p = 0.30). To our knowledge, this study is the first to report the frequency of RBW detection from osseous entrance gunshot wounds.     

PCR-DHPLC法检测硅藻SSU基因在溺死鉴定中的应用

目的采用PCR-DHPLC法检测硅藻SSU基因,评估其在溺死鉴定中的应用价值。方法 60只实验兔随机分为生前溺死（水中溺毙）、死后入水（空气栓塞致死后入水）、对照组（空气栓塞致死后不做处理）;溺死人体脏器组织;取各组织检材提取硅藻DNA,PCR扩增硅藻特异的核糖体小亚基（SSU）片段,用琼脂糖凝胶电泳检测、DHPLC检测分析。结果 6份硝酸消化法检测阴性的溺死人体器官组织检材经PCR及琼脂糖凝胶电泳检出5例阳性。生前溺死组肺、肝、肾硅藻检出率分别为100%、90%、85%,死后入水组仅肺检出硅藻（15%）,对照组各组织均为阴性;生前溺死组检出率明显高于死后入水组（P〈0.05）。10份溺死人体器官组织检材采用DHPLC法检出硅藻种类明显多于微波消解-扫描电镜法（P〈0.05）。脏器检出硅藻种类与溺死点水样一致。结论采用PCR-DHPLC法检测硅藻SSU基因,有助于溺死鉴定和溺死地点的推断,具有法医学应用价值。     

Comparison between Temperature Gradient Gel Electrophoresis of Bacterial 16S rDNA and Diatom Test for Diagnosis of Drowning

When a body is discovered in water, it is difficult to conclude whether the cause of death was drowning, even today. Although diatom testing by the digestive method is classical, we hypothesized that aquatic bacteria, as well as diatoms, might be detected in drowned bodies, and conducted temperature gradient gel electrophoresis (TGGE)‐targeting 16S rDNA. DNA was extracted from the site water, and from heart blood and liver samples from 27 bodies concluded as drowning deaths by autopsy and subjected to TGGE after amplification of 16S rDNA by polymerase chain reaction. We observed whether the feature point of each 16S rDNA from the site water and blood or liver samples matched. Considerably higher correspondence was observed in drowned bodies, and the rate was higher than that achieved with the digestive method. Moreover, TGGE is safer than the digestive method. Our study suggests that this method can aid diagnosis of drowning.     

Undetermined Manner of Death: An Autopsy Series

A manner of death may be ruled undetermined by the forensic pathologist when there is insufficient information about the circumstances surrounding the death to make a ruling. The aim of our study was to retrospectively analyze a series of autopsy cases that were classified as undetermined manner of death after complete investigations. In all, 48 cases were examined. In 23 cases (48%), the cause of death was determined. The most frequent cause of death was toxic death (n = 11). More than one manner of death was deemed conceivable for most cases (n = 39). The most frequent and the most probable manner of death was accident (n = 37). Homicide was not excluded in about 23% of the cases. Our study showed that the manner of death may remain undetermined despite an established cause of death, and even when two or more conceivable causes of death are considered. Our study pointed out that undetermined manner of death covers a wide range of situations and that homicide may be underestimated.     

东莞溺死案多发河段硅藻种群分布及其法医学意义

目的调查东莞溺死案多发河段硅藻种群分布,探讨其在溺死地点推断中的意义。方法于2009年6月至9月收集东莞市东江沙田、道滘、麻涌、石碣、石龙、茶山和企石共7个河段采样点江水样本126份,并收集2例已知落水地点溺死尸体;制作各样本硅藻涂片,计算样本间Sprensen相似系数及弦平方距离(SCD),并进行聚类分析。结果 126份水样中共鉴定出70种硅藻,其中优势度>0.02的优势种群有4种;2个案例样本分别与道滘和石碣河段样本的相似度最高,与实际落水地点基本一致。结论建立区域性溺死多发河段硅藻数据库,利用Sprensen相似系数及SCD值及聚类分析进行落水地点推断具有一定可行性。     

An Improved Method for the Diatom Test Utilizing DNA Binding Ability of Silica

In order to devise a better forensic test for diatoms, the DNA binding ability of the diatom frustule constructing by silica, in the presence of chaotropic ions were utilized. It was proved that the diatoms were able to be captured via λDNA using silica‐coated magnetic beads (Mag beads), followed by isolation and purification from the Mag beads as a solid phase by substituting the chaotropic agent with ultrapure water. Five cases of drowning, three in freshwater and two in seawater, were applied to the present method and similar results as the usual diatom test were obtained. Specimens of lung and other organs were rendered clearly visible, with elimination of foreign impurities. The present method appears applicable for detection of diatoms indirectly using PCR amplification of bound DNA or directly staining of the DNA.     

酶消化联合强酸消化法的硅藻检验CSCD

目的采用酶消化联合强酸消化法对溺死尸体的器官进行硅藻检验,并评价其应用价值。方法收集40例本地区确证为溺死的尸体,每例提取肺、肝、肾组织及现场水样,分别采用强酸消化法、酶消化联合强酸消化法、酶消化法对组织样本进行硅藻检验,从消化时间、消化能力、硅藻检出率等方面进行比较分析。结果在消化时间、消化能力方面,酶消化联合强酸消化法明显优于酶消化法;在硅藻检出率方面,酶消化联合强酸消化法明显优于强酸消化法。结论酶消化联合强酸消化法结合了强酸消化法和酶消化法的优点,并且操作安全,环境污染小,具有一定的推广和实践价值。     

Comparative and Correlation Studies of Biochemical Substances in Vitreous Humor and Synovial Fluid

Vitreous humor (VH) and synovial fluid (SF) become optional postmortem samples for biochemistry investigation. However, few studies have explored SF as a potential source, and there is still controversy whether the biochemical concentrations in bilateral VH are the same. We explored the concentrations of biochemical substances in both VH and SF, and compared between both sides and between the fluids. We also correlated the biochemical concentrations in both fluids. All samples were centrifuged after collection and the supernatant fluids were used for the analysis. The results indicated no significant difference in the constituent concentrations between the fluids from both sides (p > 0.05). Sodium, potassium, chloride, and magnesium concentrations were significantly higher in the VH, but in vice versa for glucose, uric acid, and creatinine concentrations (p < 0.001). Sodium, potassium, glucose, lactate, urea, uric acid, and creatinine concentrations between both fluids showed significant correlation (p < 0.05), but not chloride and magnesium concentrations.