| Background:The bodies recovered from water are the common types of forensic autopsy. There are lots of rivers in our country, a large amount of bodies were found every year. For example, the number of bodies found in water in Guangzhou was nearly a thousand per year. A body found in the water most belong to accidental drowning, suicide or homicide were the possible causes of death of a body found in the water. The judgment of antemortem or postmortem drowning is the key to the cases investigation. The mechanism of drowning death is that the liquid inhaled into the lungs prevents gas exchange, leads to hypoxemia, carbon dioxide retention and irreversible brain hypoxia. For fresh bodies found in water, snout mushroom-like froth, aqueous emphysema, silt in the respiratory tracts are the supportive evidences for drowning. The Pearl River is located in the region of subtropical climate. According to the reports,87%of the bodies found in the water were highly corrupted without typical signs of drowning. Those become an important problem in forensic science.Diatoms are unicellular having a cell wall made of silicon dioxide. The glass frustule is composed of two valves, which fit together with the help of a cingulum, or set of girdle bands. There are about16,000species and174genera of diatoms reported which are having different shapes and sizes varying from1to200μm. Most diatoms exist singly, although some join to form colonies. They are found in both fresh and salt water, in moist soil, and also on the moist surface of plants. The cell walls of diatoms with high resisting ability were difficult to destroy, even digesting with concentrated sulfuric acid, nitric acid and high temperature. They would be remained after digestion, and very beneficial to diagnosis of drowning.For last100years after Revenstorf found diatoms in the lung tissues in1960, many scientists have employed various extraction methods individually and in combinations in order to isolate diatoms from water and tissues samples successfully. Analysis of diatoms present in the body tissues like lungs, liver, spleen, blood and bone marrow has been undertaken as supportive evidence in downing cases.Diatoms can lead not only to the determination of cause of death but also the suspected site of drowning, if analyzed quantitatively and qualitatively.Until now, a lot of diatom test methods have been developed for the diagnosis of drowning since the first description of diatoms in drowning. The most common extraction technique consists of chemical digestion by nitric or sulfuric acid, soluene-350, or enzymes. The conventional acid digestion method consisted of acid digestion, centrifugation, and observation by light microscopy. The diatom extraction from tissues is an important step in the whole method.Sampling and digestingNitric acid digestion method was first introduced by Kasparek in1937, laid the foundation of strong acid digestion method. In forensic practice, methods with the nitric acid in sand bath, nitric acid, nitric acid and hydrogen peroxide, sulfuric acid and copper sulfate, nitric acid and ethanol were developed. But there were some disadvantages in the process of strong acid digestion method, such as high intensity, long time digestion, risking of burning, nitrogen dioxide pollution and diatom destruction. Soluene-350was used by Hiroshi to digest tissues with advantage of time-saving, simple operation. But the ability of digestion is not good enough, and the reagent is difficult to get. Therefore, it is not suitable for application in forensic practice. In addition, the enzyme digestion method, microwave digestion using nitric acid and silica gel gradient method were used in forensic diatom test. But the disadvantages of long time digestion, poor ability of digestion, little tissue quantity of each digestion were existed in all these methods.EnrichmentCentrifugation is generally used in forensic science laboratories around the world to enrich diatoms. The standard forensic diatom test method in China says: centrifugate at the speed of4000r/min for15min, discard the supernatant, centrifugate after adding redistilled water. Repeat the above steps until the Ph value of the solution is6.5-7.5. Zhao Jian etc. found that at the speed of4000r/min for15min, the rate of diatom loss is between29.94%to49.60%(34.18±9.19%), a large number of diatoms abandoned with the supernatant.ObservationLight microscopy was most widely accepted in forensic laboratory in the world. Some mini diatoms are easy to be omitted because of restriction of magnification. And the ornamentation on diatom cell surface is difficult to identify. In addition, the reason of the date of diatoms in the test range widely is the magnification of light microscopy according to some researchers. Therefore the accuracy of the light microscopy observation, especially the accuracy of the diatom classification needs to be further developed.Torre etc. and Pachar etc. have reported with traditional acid digestion and scanning electron microscopy (SEM) detection of diatom. Comparing with the light microscopy, SEM is with the advantages of high magnification and large depth of field, can observe the fine structure of the diatoms, and easy to store emages for identification of diatom species.Due to limitations of the existing diatom test methods, the diatom test in the death investigation of bodies found in water can not work usefully. And because of the lack of standard diatom test method, there is lot of controversy about the diatom test. Because of difficult operation with samples from decomposed corpses, low positive rate, easy to be polluted, harmful to operator’s health, the forensic medical workers urge to develop new methods, new technology for diatom test.Objective:1. Establish accurate and efficient forensic diatom test method;2. Compile the atlas of diatom species in the Pear River in Guangdong province, and offer a reference for forensic workers to identify the diatomsMethods:1. The establishment of the Lefort aqua regia digestion method:1)22white rabbits were randomly divided into two groups, the Lefort aqua regia digestion group (n=10), the traditional acid digestion group (n=10), the control group (n=2).1L water sample taken from the pearl river in Guangzhou city (the water sample was fixed with formalin solution and keep in dark place);2) The two kidneys of rabbits were prepared and about2g kidney tissues were sampled and cut into pieces in the100ml beaker;3) The steps of the Lefort aqua regia digestion method:2ml water sample were added;15ml concentrated nitric acid and5ml of concentrated hydrochloric acid were added in the breaker;2ml of hydrogen peroxide were dropped into at the speed of1drop every3seconds; Samples were left without interruption for15min; Samples were put into the thermostatic water tank at85℃for50min, and during heating, another3ml of hydrogen peroxide was added at the rate of1drop per3s.4) Steps of traditional acid digestion method:2ml water sample were added;20ml concentrated nitric acid and5ml of hydrogen peroxide were added in the breaker; Samples were put into the thermostatic water tank at85℃for50min.5) The digestion solution was filtered with a millipore filtration (Pore diameter size is0.45μm). Then the membranes were analyzed by SEM after the filter membranes were coated with a layer of gold.6) Values in the text and table were expressed as the means±SD, and statistical analyses were performed by two-way analysis of t-test using SPSS17.0for windows. P<0.05was considered as statistically significant. 2. The Lefort aqua regia digestion method detecting diatom in rabbits’s organs1)44white rabbits were randomly divided into three groups:drowning group (n=20,10for the Lefort aqua regia digestion-membrane filtrition-SEM observation method, another10for the traditional acid digestion-centrifugation-light microscopy observation method), post-mortem immersion group (n=20,10for the Lefort aqua regia digestion-membrane filtrition-SEM observation method, another10for traditional acid digestion-centrifugation-light microscopy observation method), control group (n=4).2) The establishment of the animal model:The animal model of drowning (the rabbits were immersed in0.5m under water for1min, and then breath for30s. Repeat the steps until the white rabbit dead. Keep in the water for30min); The animal model of post-mortem immersion (The rabbits were killed directly through air embolism. Keep in the water for30min.); The animal model of non-drowning (The rabbits were killed directly through air embolism.).3) The rabbits liver, lungs and kidneys were analyzed. The time of digestion, detection rate, numbers of diatoms and observation were recorded and statistically analyzed.3. The Lefort aqua regia digestion method detecting diatom in Pear River1)19different sampling sites (6in the West River,9in the North River, and4in the East River) were sampled by us in June and September2012.%2) Water samples from each sampling site was digested and then observed by scanning electron microscopy. The diatom genera found in samples were recorded.Results:1. The degree of digestion of the kidney tissues in the limited time (50min) was evaluated by the weight of the precipitate on the filtration membrane after digestion. The weight of the precipitate of the Lefort aqua regia digestion method was0.00604±0.00310g/2g, and that of conventional acid digestion method was0.00979±0.00204g/2g. The digestive capability of Lefort aqua regia digestion method was superior to conventional acid digestion method, with the difference being statistically significant (P<0.01) 2. There was a minimal amount of organic matter in the samples digested with the Lefort aqua regia, and the structure of diatoms remained intact with a clear background and little impurity in all the replicates performed. The fragment of diatoms was rarely found. The results indicated that the digestive ability of Lefort aqua regia digestion method was very strong, and fewer residues were left.3. There was no diatom found in the negative control sample. The standard number of diatoms in water samples was711.2/m. The recovery of diatoms with the Lefort aqua regia digestion method was71.1±10.3%, and that of conventional acid digestion method was66.3±9.0%. There was no statistical difference between the two groups (P>0.05). The results showed that there was no more diatom loss in the Lefort aqua regia solution.4. The consuming time of the Lefort aqua regia digestion-membrane filtrition-SEM observation method was less than the the traditional acid digestion-centrifugation-light microscopy observation method, about1/4in digesting lung tissues,1/3in liver tissues and1/3in kidney tissues.5. The consuming time of digesting different tissues with the Lefort aqua regia digestion-membrane filtrition-SEM observation method and the the traditional acid digestion-centrifugation-light microscopy observation method was as follows: lung<liver<kidney. Lung tissues were much easier to digest than livers, and kidney tissues is the most difficult to digest.6. There was no diatom found in the negative control sample in the lung, liver, kidney tissue in the control group. The results of the Lefort aqua regia digestion membrane filtrition-SEM observation method were positive in all samples of drowning group while3cases of liver tissues and2cases of kidney tissues were negative in the traditional acid digestion-centrifugation-light microscopy observation method. There was no statistical difference between the two methods in detecting diatoms in lung, livers and kidney tissues (P>0.05).3cases of lung tissues were positive in the Lefort aqua regia digestion-membrane filtrition-SEM observation method of post-mortem immersion group while1cases of lung tissues were positive in the traditional acid digestion-centrifugation-light microscopy observation method. The results of liver tissues and kidney tissues were negative.7. In the drowning group, the diatoms numbers detected by the Lefort aqua regia digestion-membrane filtrition-SEM observation method was more than the the traditional acid digestion-centrifugation-light microscopy observation method, about10times in digesting lung tissues,4in liver tissues and6in kidney tissues. In the post-mortem immersion group, the diatoms numbers of3positive cases detected by the Lefort aqua regia digestion-membrane filtrition-SEM observation method were12/g,15/g,13/g. And the diatoms numbers of1positive cases detected by the the traditional acid digestion-centrifugation-light microscopy observation method were2/g.8. The light microscopy scanned the fields in a laborious manual process inefficiently. The fine structure of diatoms was difficult to observe. The SEM system scanned the fields automatically, and the picture of each field was taken, and we saved them in the computer. The fine structure of diatoms was easy to observe.9.21diatom genera, including Achnanthes, Cocconeis, Coscinodiscus, Cyclotella, Cymatopleura, Cymbella, Diatoma, Diploneis, Gomphonema, Gyrosigma, Hantzschia, Melosira, Navicula, Nitzschia, Pinnularia, Rhoicosphenia, Stauroneis, Stephanodisus, Surirella, Synedra, Tabellaria, were found in all the samples.Conclusion:1. The Lefort aqua regia digestion method for diatom extraction has been developed and tested. Diatoms were found in our study, and the new method was helpful for diatom identification. The digestive capability of Lefort aqua regia digestion method was superior to conventional acid digestion method; the structure of diatom remained almost intact; and the recovery of diatom was comparable to the conventional acid digestion method. In conclusion, the Lefort aqua regia digestion method is an improvement over the conventional acid digestion for recovery of diatoms from tissue samples.2. Hundreds of diatoms pictures were taken by SEM, it would be a valuable reference of diatoms identification for forensic experts. |
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