硅藻检验方法综述

水中尸体的调查在法医学检案中占了重要的一部分。水中尸体未必就意味着其死亡原因是溺死,也可以由其他原因引起,例如死后入水。实际上,尸体现象很难证明死亡的原因。所以,关于受害者是否为"真的"溺死的问题常常不容易解决。目前,硅藻检验被认为是溺死诊断的"金标准"。通过硅藻的定性定量分析,不仅可以对死亡原因有更直接的判断,也可以指向可能的溺死地点。本文介绍国内外硅藻检验的样本采集、检验方法、结果评价的研究进展,并对硅藻检验研究进行展望。     

鉴定溺死的实验研究——荧光分光光度计检测肺组织浮游生物叶绿素(a)

本实验应用荧光分光光度计检测肺组织浮游生物叶绿素(a),以期通过准确的定性、定量研究来诊断溺死。实验性研究表明本法准确可靠,选择性强、灵敏度高,不受腐败及污染等因素影响,亦不存在含叶绿素(a)浮游生物生前进入并存在于人体器官的问题。同法对2例水中尸体进行了检测,结果与实验性研究相符、提示本法可应用于实际检案。     

溺死鉴定中硅藻检验的应用

在法医学检案中,溺死诊断以及入水地点的判定一直是重点和难点之一,硅藻检验被认为是一种相对可靠的诊断溺死的方法,而根据硅藻的群落特征推断其入水区域具有一定的可信度。对不同水域内硅藻进行研究,可为溺死鉴定及入水区域的判定提供参考。本文就硅藻相关的生物学特性、检验方法等相关的国内外研究进展进行综述,供法医学工作者在相关科研和检案实践中参考。     

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.     

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.     

硅藻定量检验和种属鉴定的实验研究

本实验用家兔18只,分为溺死组、死后(抛尸)入水组及对照组,每组各6只。采用消化—光镜法作肺、肝、肾硅藻计数(定量)及种属鉴定检验,同时作水样检验及空白对照。结果:(一)进一步证实生前硅藻可经空气源性进入肺脏并存积于大循环内脏(肝、肾)的概念;(二)提示水中尸体肺脏如有大量硅藻检出,仅此即可作为诊断溺死的可靠依据;空气源性吸入及检验过程中的污染并不影响该检验结果对鉴定溺死的判断。种属分析溺死组肺与水样的主要硅藻种属相符,而肝、肾中硅藻计数及种属分析均无诊断意义。     

Prevalence of diatom frustules in non-vegetarian foodstuffs and its implications in interpreting identification of diatom frustules in drowning cases

Detection of diatom frustules in bone marrow (diatom test) is used for diagnosing ante-mortem drowning where the usual signs of drowning are not present in dead bodies recovered from water. However, controversies over the reliability of diatom test results are continuing. There have been indications on the possibilities of diatoms entering into systemic circulation from atmospheric air, food and drink. While diatoms have been demonstrated in the gut content of edible marine forms such as shrimps and clams, the present study, for the first time, provides empirical evidence on the prevalence as well as abundance of diatom frustules in the samples of cooked non-vegetarian foodstuffs that impend human consumption in Kelantan, Malaysia. It is found that 50 g each of cleaned and cooked prawns and of clams impending human consumption contain about 8360 and 29,054 diatom frustules, respectively. A person accustomed to prawn and clam food would be ingesting an estimated 2 million diatoms in a single year. Considering the suggestion that detection of five diatom frustules in 10 g of bone marrow would suffice for concluding drowning as mode of death, and the fact that there is yet no proof that diatom frustules do not enter into the human systemic circulation through the digestive tract, the estimated number of diatom frustules routinely ingested acquires significance since entry of a few of such ingested frustules into the systemic circulation can lead to false positive test results. The findings of this research raise two important issues: first, population based routine food related diatom ingestion requires to be estimated, and, second, studies have to be initiated to categorically prove or disprove the possibility of entry of diatom frustules into the systemic circulation via the digestive tract.     

Study of the diagnostic value of iron in fresh water drowning

The aim of our study was to test the diagnostic value of iron (Fe) in fresh water drowning by investigating the postmortem levels of hemodilution in drowning cases compared to control cases. Twenty-six typical fresh water drowning cases were selected from 128 immersion cases autopsied in our Department of Forensic Pathology between 1998 and 2004. The exclusion criteria were a long postmortem interval and other causes of death than drowning. For all selected cases, the diagnosis of drowning was based on the presence of autopsy findings and positive diatom test. A control population of 12 cases was also selected. For each drowning and control case, iron blood levels were measured in the left ventricle (LV) and right ventricle (RV) of the heart. The mean difference of iron concentration (RVFe-LVFe) between the drowning group and the control group was statistically compared. Furthermore, iron measurements were performed in 19 drowning cases showing advanced putrefaction. The mean difference of iron concentration was significantly higher in the drowning cases compared with controls (P<0.001). All drowning cases showed hemodilution. No overlap was found in the RVFe-LVFe levels between the two groups. Resuscitation attempts seemed to have no effect on the results. In cases of drowning showing advanced putrefaction, the iron test was not reliable because biochemical iron measurement was often prevented by no sufficient blood in the heart or postmortem clots. In conclusion, according to our results, iron seems to be a good biochemical marker in fresh water drowning with a short postmortem interval.     

Limbs found in water: investigation using anthropological analysis and the diatom test

We report the investigation, using a multi-disciplinary approach, of five cases of dismembered limbs which were recovered from Lake Ontario, Lake Erie and the Niagara River, and examined at the Office of the Chief Coroner for Ontario. In all cases, postmortem examination revealed that the limbs had been disarticulated in the postmortem period, by non-human taphonomic processes. In addition to routine gross examination, the femur and/or tibia were assessed using anthropological methods to give estimates of the sex, age, race and stature of the individual. The anthropologic data facilitated the identification of one of the cases. In all cases, nitric acid extracts of the femoral bone marrow were prepared and examined for the presence of diatoms. In all instances, diatom frustules were recovered from marrow extracts, indicating that drowning was the cause of death or at least a significant contributing factor in the cause of death. The use of the diatom test was helpful in excluding the possibility that the limbs were dismembered from individuals who had died by means other than drowning, and had been subsequently 'dumped' into water. The application of anthropological methods and the diatom test for drowning may significantly enhance the medico-legal investigation of body parts recovered from water, and we present an overview of useful techniques here. Anthropological data may facilitate identification, and the diatom test may establish a cause of death.     

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。     

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。     

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。     

溺死的法医学检验现状及进展

本文概括阐述了近年来,国内外学者将硅藻的检验、内脏异物颗粒和微量元素成分的检验、血液化学的检验、电子显微镜的观察等方法应用于溺死的诊断,试图为溺死的法医学鉴定提供准确的诊断指标。     

测定血锶含量鉴定溺死的法医学意义

为评价血锶含量测定对诊断鉴定溺死的实用价值,作者用家兔进行实验,分别测定溺死与死后入水家兔左心血中锶含量。结果证实:1.死后较短时间内血锶含量测定确可鉴定溺死;2、死后24小时内仅可帮助鉴定溺死。实验表明水中浸泡时间因素对血锶含量确有显著影响。两例实际检案的应用观察,较实验性研究为佳,值得进一步研究。     

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。     

微波消解-扫描电镜联用检测脏器内硅藻

目的介绍一种微波消解-扫描电镜联用检测脏器内硅藻的新方法。方法大白兔30只,随机分为生前溺死组（n=15）和死后溺尸组（n=15）。提取兔肺、肝、肾、股骨骨髓和现场珠江水样,采用建立的微波消解-扫描电镜联用法检测脏器组织和水样中的硅藻,并与硝酸乙醇消解-扫描电镜联用的方法进行比较。结果生前溺死组的兔肺、肝、肾、股骨骨髓组织中大多数观测到与现场珠江水样一致的硅藻,微波消解-扫描电镜联用法检测脏器内硅藻的平均含量分别为：2505.2个/2g肺,18.7个/10g肝,6.5个/10g肾,6.3个/0.5g骨髓;肺与肝、肾、骨髓检出硅藻的阳性比例为86.7%,脏器硅藻检出含量和硅藻检验阳性比例均明显高于硝酸乙醇消解-扫描电镜联用法所测值。死后溺尸组的兔脏器组织均未观测到硅藻。结论微波消解-扫描电镜联用法检测脏器内硅藻,高效、安全、环保,硅藻检验灵敏度高,降低了劳动强度,提高了定性定量分析准确度,且能有效避免污染,在法医学溺死鉴定中具有良好的应用价值。     

The forensic medical evaluation of the results of determining diatomaceous plankton in the diagnosis of drowning

Evaluation of demonstrative value of method based on diatom plankton determination as a diagnostical sign of death due to drowning was performed. Plankton was detected in the viscera in 37% of cases (as a whole 376 bodies of people who drowned in fresh water were examined). Plankton was detected in aspiration type of drowning and its mixed variant. In spastic and reflector types of drowning plankton wasn't detected. According to author's opinion this method is an objective diagnostical test to determine the cause of death of people when their bodies are found in water.     

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。     

溺死诊断中的浮游生物检测法

溺水是常见的死亡原因之一,浮游生物检验是法医学鉴定溺死的重要依据。浮游生物检验方法可分为破机法、非破机法和分子生物学方法三大类。前两者主要以形态标记为检验对象,依赖于浮游生物的理化特性,有很大局限性。分子生物学方法以分子标记为检验对象,适用范围广,信息丰富,是值得探索的新方法。本文回顾溺死鉴定中的各种浮游生物检测法,供法医工作者在实践和研究中进行参考。     

A study on the diagnosis of drowning by examination of lung chlorophyll(a) of planktons with a spectrofluorophotometer.

An investigation on the diagnosis of drowning by analysis of chlorophyll(a) (Chl.(a)) of planktons in the lung sample with a spectrofluorophotometer (SFPM) is reported. The diagnostic criteria or main points of death by drowning are suggested are according to our experimental studies. Meanwhile two cases of submersion in water were investigated and the causes of death were diagnosed correctly. It is concluded that examination of lung Chl.(a) of planktons could be used as a reliable diagnostic method in forensic practice.