溺死法医学鉴定的研究新进展

综述了近年来在硅藻检验、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等情况。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。     

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。     

碎浆法联合硝酸破机法检验硅藻

硅藻检验是法医病理学用于鉴定溺死的传统方法,尤其对高度腐败和白骨化尸体具有重要意义。目前,硅藻检验的破机法主要有：强酸法、物理（机械、焚烧）法、生物酶消化法等。本文尝试采用控温高速组织捣碎机（江苏江阴科研仪器制作厂）制成组织匀浆,以硝酸破机法进行硅藻检验,取得较好结果。     

破机罐的研制

本文介绍一种检验硅藻时破坏有机质(简称破机罐)的工具。对于溺死案件的法医学鉴定、教学及法医工作者的劳动保护均具有重要的意义。 硅藻检验是鉴别生前溺水死亡与死后抛尸入水的一种可靠、实用的方法。特别是在尸体高度腐败或白骨化时,甚至是唯一的方法。在检验器官组织中的硅藻时要先破机,为此,我们成功研制了破机罐,为硅藻检验提供了一种理想的破机工具。     

两种硅藻检验方法的比较

目的比较标准硝酸破机法和微波消解-扫描电镜联用法检验水中硅藻的效果。方法采集广州市区海珠桥段珠江水样本,分别采用标准硝酸破机法(GA/T 813-2008)和微波消解-扫描电镜联用法进行检验,比较两种方法对硅藻的检出率;再将水样本按25、50、100、200倍稀释,分别采用两种方法检验,比较其最低检出量。结果微波消解-扫描电镜联用法与标准硝酸破机法硅藻检出数量和检出率比较,前者明显高于后者,差异具有显著意义(P0.01),标准硝酸破机法硅藻最低检出量为37.2~18.6个/m L,微波消解-扫描电镜联用法则低于18.6个/m L,在样本稀释200倍时两组之间的差异具有显著意义(P0.01)。且经微波消解后硅藻结构未产生明显变化。结论微波消解-扫描电镜联用法对硅藻破坏程度低,检出率高,在法医学溺死案件中具有良好的应用价值。     

硝酸乙醚破机法检测硅藻的研究

脏器中检出硅藻是证明生前溺死的可靠方法.长期以来,法医实验室一直沿用硝酸沙浴法检验硅藻.八三年有人报道采用硝酸酒精法破机,在许多方面优于硝酸沙浴法.本文采用硝酸乙醚法破机,取得了较好的结果.现报道如下.     

改良硝酸破机法检验尸体组织中的硅藻

目的 探讨改良硝酸破机法与传统硅藻检验方法的差异以及优点.方法 无水乙醇充分固定尸体组织,取材80～100g,剪碎后使用硝酸破机,离心水洗去除有机成分后涂片镜检. 结果硅藻纹理清晰,容易辨识,方便记数及种类甄别.结论 改良硝酸破机法检验硅藻操作简单,检出率较高,标准化的实验过程可有效避免污染对实验结果的影响,提高实验结果的稳定性,避免实验过程中对人员和环境的危害,增加实验过程的安全性.     

硅藻硝酸消化法与浮游生物16S rDNA PCR法在溺死鉴定中的比较

目的比较和探讨硅藻硝酸消化法与浮游生物16S rDNA PCR法在溺死鉴定中的应用价值。方法收集40例温州医科大学法医学系2010—2011年受理并证实溺死的鉴定案件,每例案件标本包括肺、肾、肝及现场水样4份样本,分别运用硅藻硝酸消化法与浮游生物16S rDNA PCR法对标本进行检验,硅藻硝酸消化法和浮游生物16S rDNA PCR法所需各器官检材量分别为约20g和2g,现场水样分别为15mL和1.5mL,从所需检验时间以及检出率等方面进行比较分析。结果硅藻硝酸消化法检出硅藻主要为中心硅藻纲和羽纹硅藻纲,浮游生物16S rDNA PCR法可扩增出一条162 bp的条带。平均检验每例案件所需的检验时间,硅藻硝酸消化法为(95.30±2.78)min,少于浮游生物16S rDNA PCR法(325.33±14.18)min(P0.05)。两种方法对现场水样及肺样本的检出率均为100%,而对肝及肾样本的检出率,浮游生物16S rDNA PCR法均为80%,高于硅藻硝酸消化法40%和30%(P0.05)。结论在溺死的法医学鉴定中,应根据具体情况来挑选合适的实验室检验方法。与硅藻硝酸消化法相比,浮游生物16S rDNA PCR法具有检材用量少、信息量大、特异性高等特点,有一定的推广和实践价值。     

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。     

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。     

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.     

A Simple Digestion Method with a Lefort Aqua Regia Solution for Diatom Extraction

Presence of diatoms in tissues has been considered as a significant sign of drowning. However, there are limitations in the present extraction methods. We developed a new digestion method using the Lefort aqua regia solution (3:1 nitric acid to hydrochloric acid) for diatom extraction and evaluated the digestive capability, diatom destruction, and diatoms' recovery of this new method. The kidney tissues from rabbit mixed with water rich in diatoms were treated by the Lefort aqua regia digestion method (n = 10) and the conventional acid digestion method (n = 10). The results showed that the digestive capability of Lefort aqua regia digestion method was superior to conventional acid digestion method (p < 0.01); the structure of diatom remained almost intact; and the recovery of diatom was comparable to the conventional acid digestion method (p > 0.05). The Lefort aqua regia reagent is an improvement over the conventional acid digestion for recovery of diatoms from tissue samples.     

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。     

Diatom test with Soluene-350 to diagnose drowning in sea water.

The authors tested the Soluene-350 method to detect diatoms in three cases of sea water drowning. The negative results obtained in all three cases prompted us to re-test the method on samples of fresh water and sea water microalgae. The experiment showed that the siliceous frustule of sea water diatoms is solubilized by Soluene-350 while that of fresh water diatoms is resistant to the treatment. The method, which is effective in cases of drowning in fresh water, should therefore be used with caution in sea water drowning.     

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.     

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。