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。     

京杭大运河济宁段秋冬季硅藻分布及法医学应用

目的通过对京杭大运河济宁段及济宁周边水域秋冬两季水样中硅藻种类及数量的分析,寻找不同落水地点之间的差异。方法在A(6)、B(6)、C(3)三条河流中选取15个站位点,分别在秋冬两季进行水样采集,采用石墨消解-扫描电镜法对各站位点的硅藻进行定性和定量分析。结果秋冬两季节各站位点硅藻含量间差异有统计学意义(P0.05)。不同站位点之间优势种属的种类和个数不完全相同,且相同优势种属间优势率完全不同,因此各站位点之间存在种属优势率差异。C河流硅藻数量(每视野 100个)明显多于A、B河流硅藻数量(每视野15个),A、B河流硅藻数量与C河流硅藻数量相差约10倍,硅藻数量上存在显著差异。结论对于溺亡案件的法医学分析,在落水时间和落水地点上提供了一定的参考。     

肺组织硅藻定量分析在实验动物溺死诊断中的应用

目的探讨肺组织硅藻检验在法医学溺死诊断中的应用价值。方法将实验动物随机分为溺死组、死后入水组和陆地死亡组,采用微波消解-真空抽滤-扫描电镜联用的硅藻检验方法对肺组织和溺液硅藻进行定量分析,记录肺组织与溺液硅藻含量的比值(ratio of content of diatoms in lung tissue and drowning fluid,C_L/C_D)。结果溺死组实验兔的C_L/C_D值(5.82±3.50)高于死后入水组(0.47±0.35);溺死组实验猪肺叶不同部位的C_L/C_D值均高于死后入水组;在海水、咸淡水交界、河道淡水及湖泊淡水中,溺死组实验猪的C_L/C_D值均高于死后入水组(P0.05)。动物实验中,C_L/C_D值1.6的案例均来自溺死组。结论 C_L/C_D值是溺死诊断中有较好应用前景的指标。     

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(P0.01),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。     

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。     

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。     

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.     

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。     

沈阳市内浑河河段中硅藻数量及种群变化规律

目的观测沈阳市内浑河河段中硅藻数量及种群的变化规律,为法医学溺死鉴定及判断落水地点提供技术帮助与科学证据。方法 2011年全年对沈阳市内浑河河段布观测点,每月采集水样并观测硅藻数量及种群的变化规律,每周采集水样并观测硅藻优势种群的变化规律。结果沈阳市内浑河河段中硅藻数量、种群及优势种群均随时间、地点而变化;硅藻数量及种群数12~2月最低,之后逐渐升高,5月达高峰,略有下降10月达第二高峰期,之后迅速减少;硅藻优势种群4~11月同一采样点相邻两周间变化明显,7~8月同一周内不同采样点变化不明显。结论河水中硅藻变化规律复杂,受环境、水文等多种因素影响,在法医学溺死鉴定及落水地点推断过程中,应充分考虑硅藻数量及种群的变化规律。     

成都市主城区水中尸体多发河流区段硅藻分布

目的研究成都市主城区水中尸体多发河流区段所含硅藻的种属分布。方法采集2014年10月成都市(锦江区、金牛区、青羊区、武侯区、成华区)5条河流区段共39个采水点水样标本,制作硅藻涂片后运用生物显微镜及数码显微采集系统对水样中硅藻种属分布及构成比进行研究。结果成都主城区所检水域共检见硅藻种属21种,各水域硅藻优势种属及构成比存在差异。各河流区段上、中、下游采样点均存在差异藻属。结论初步建立了成都主城区河流区域性硅藻种属图谱及区段性种属分布构成比数据库,对分析评价硅藻检测在案件侦查中推断落水点具有特殊意义。     

Colonization of diatoms in and on porcine bone substrate and considerations of diatom ecology for forensic science

The presence of diatom algae in bone marrow has been used as forensic evidence of drowning for several decades; however, these studies are based on known or suspected recent drowning events. This study addresses the potential for diatoms to enter the bone marrow of skeletal remains, that is, de-fleshed long bones post-mortem. In laboratory and field experiments, bones were either inflicted with two access points by a cut and acid pitting or left intact. The bones were submerged in water for at least 1 week and up to 3 months. Samples of the bone surface and marrow were inspected for diatoms. The analysis considered the time required for diatoms to enter marrow and whether genus characteristics like size or mobility affect entry. The presence of an access point influenced diatom entry in that bones without an introduced access point had zero to one diatom present in the marrow, whereas a bone with an access point had >150 diatoms present in the marrow. The results of both laboratory and field phases suggest that diatoms will reliably colonize bone in as quickly as 1 week, establishing and maintaining communities for at least 3 months. However, the bone surface assemblages differ from the source community. Bone marrow displayed even more restrictive access to diatom colonization, resulting in communities dominated by small raphid diatoms. Based on these findings, we suggest some caveats on the use of diatoms as trace evidence in forensic science with recommendations for future avenues of research.     

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。     

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。     

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.     

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.     

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.     

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.     

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.     

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.