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.     

Diatoms in forensic expertise of drowning--a Macedonian experience

Results obtained by examination of 22 human cases suspected for drowning, one human case of death other than drowning and several tests on laboratory rats were used as a basis for evaluation of diatom method as supportive in forensic expertise of drowning. The recovery of diatoms from various examined organs, their qualitative and quantitative composition, if properly treated without the possibility of contamination, can be a reliable proof of the time and place of drowning. The priority of organ examination (external microflora determination, lungs, brain, heart (and/or blood), stomach, liver and kidney, and finally bone marrow) is discussed and established as well as the basic future research on cases suspected of drowning, but also on non-drowned victims and laboratory animals.     

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.     

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.     

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.     

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。     

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.     

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。     

辽宁省主要河流冰封期及开河后浮游硅藻变化规律

目的观察分析辽宁3条河流中浮游硅藻数量及种群构成在冰封期及开河后的变化规律,探讨其在法医学应用中的意义。方法于2012年1～4月中旬分别在辽河、浑河沈阳段及太子河本溪段布点取水,逐月观测硅藻数量及种群构成的变化规律,计算3条河段各月水样中硅藻数量平均值（个／10mL）以及主要硅藻属种及其构成比。结果3条河流冰封期及开河后硅藻种属数量和构成比均有明显变化。在冰封期（1～3月）硅藻种属数量趋于稳定,中心纲硅藻为优势种群,仅见少量羽纹纲硅藻,各月之间硅藻数量变化缺乏统计学差异;初春开河后（4月）硅藻种属数量显著增加,与冰封期差异明显（P〈0．01）,中心纲及羽纹纲硅藻混杂存在。结论辽宁主要河流在冰封期及开河后水中硅藻种群构成及数量变化大,春季水中浮尸的死因鉴定及落水点判断应合理分析现场水样与脏器中硅藻种群及数量差异。     

Detection of diatoms in bone marrow (femur) of nondrowned (author's transl)

Diatoms in the bone marrow (femur) of 16 nondrowned bodies were detected by a modern method of ultrafiltration. The rate of diatoms were found to be in the same range as in cases of drowning. The results deny the proof of diatoms even in bone marrow to be useful any longer for the diagnosis of death from 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。     

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。     

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.     

上海地区水域中硅藻的基本分布调查

在法医检案中,水中尸体的检验是工作中的难点。硅藻检验作为水中尸体检验的必须手段,因其操作简便,被广泛应用。通过实际案例,对上海地区水样中硅藻的分类、分布进行了疏理。以期为上海地区溺水死亡案件落水地点判断提供参考。     

辽宁清河、棋盘山两座水库中硅藻种群及数量变化规律的法医学研究

目的研究辽宁清河、棋盘山两座中小型水库中硅藻种群及数量可能存在的区域性和时间性差异,拟为法医相关检案明确死因、规范现场水样提取方法及正确分析落水地点提供科学依据。方法选择辽宁境内棋盘山秀湖和铁岭清河两座水库作为研究对象,2012年春季（5月）及夏季（8月）分别在水库最宽段从岸边向水库中心以一定间距设点取样,观察硅藻种群及数量的变化。结果硅藻数量由岸边向水库中心呈现最低-增多-最多-减少-稳定的变化趋势,岸边硅藻数量最少,近岸带硅藻数量达峰值,之后硅藻数量降低,经一段水域过渡之后在水库中心区域硅藻数量趋于稳定;同一观测点春季硅藻数量明显多于夏季;岸边及近岸带以羽纹纲硅藻为主,在过渡区域,中心纲硅藻与羽纹纲硅藻混杂出现,水库中心区域内中心纲硅藻占绝对优势。结论水库型静水水体中,硅藻种群和数量存在区域性及时间性差异,合理布点取水,对于比对和分析现场水样与死者脏器中硅藻种群及数量至关重要。     

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。     

水中尸体PMCT最新研究进展

溺水是公安法医日常检案中最常见的死因之一,水中发现的尸体是否为生前入水,是公安法医日常鉴定的要点也是难点,而一旦溺亡的案件中掺杂了其他复杂的案情,其鉴定意见失真也极易引起不良的社会影响。法医鉴定过程中,溺死一般依据尸体征象、组织病理学检验和硅藻检验等综合考虑。PMCT为法医诊断溺死提供了新的思路并被许多国家和地区常规使用于诊断溺死。本文综述了国内外近年来使用PMCT对溺死尸体呼吸系统、消化系统和循环系统等方面的研究成果,讨论了PMCT诊断溺死的应用价值与潜力,并对国内PMCT发展的必要性和前景进行了展望。     

Diatoms and drowning--once more

The content of diatoms in 5 samples: lung-, kidney- and liver-tissue plus columna- and femur-marrow from each of four drowned and four non-drowned persons has been investigated. Diatom valves were found in all the samples. It seems, however, impossible to point out any characteristic differences between the composition of the diatom 'flora' in drowned and non-drowned persons. Consequently it will not be possible by means of diatoms to prove that a person died by drowning.     

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。