Bioluminescent Assay of Microbial ATP in Postmortem Tissues for the Estimation of Postmortem Interval

To study the relationship between changes of microbial ATP in four kinds of murine tis-sues and the postmortem interval （PMI）, healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals. The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentra-tion of microbial ATP in muscle increased with PMI time. The peak appeared at the 7th day after death, and at the 10th day, microbial ATP in muscle tissue increased again. In internal organs, the peaks of microbial ATP were observed at the 8th day after death and the level decreased during 8–10 d. The differences in microbial ATP concentration in liver, spleen and kidney were not statistically significant. During day 0 to day 9 after death, the correlation was best between PMI and microbial ATP in muscle. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.02X3–0.166X2–0.666X＋13.412 （R2=0.989, P〈0.01）. In internal organs, the best correlation was found between PMI and microbial ATP during day 0 to day 10. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.016X3–0.127X2–0.809X＋13.324 （R2=0.986, P〈0.01）. There existed high correlations between PMI and microbial ATP concentration in rat tissues. Since only a small amount of tissue was needed for the detection and the sample was not affected by self-decomposition, the method may extend the time range of PMI estimation.

Spectrophotometric Determination of Trimethylamine-nitrogen in Cadaver Tissues for the Estimation of Late Postmortem Interval:A Pilot Study

To study the relationship between the late postmortem interval （PMI） and trimethyl-amine-nitrogen （TMA-N） in postmortem tissues of cadaver, TMA-N in muscles, livers and kidneys of rats was measured at different postmortem intervals （PMI） by using a modified spectrophotometric method. The results indicated that the detection sensitivity of TMA-N was 1 mg/L, and there was a good linear correlation between the value of absorbance （A value） and TMA-N at the concentration of 1-10 mg/L （R2=0.9991）. Although TMA variation in muscles was different from that in inner organs during the time since death, TMA-N changes in cadaver tissues was positively correlated with PMI. During 2 to 7d since death, the best correlation between PMI and TMA-N concentration was found in muscles. With PMI as an independent variable, the cubic polynomial regression equation was y=-0.457x3 ＋6.519x2-24.574x＋27.207 （R2=0.969）. During 3 to 8 days since death, PMI was best correlated with TMA-N concentration in inner organs. With PMI as the independent variable, the cubic polynomial regression equation was y=0.509x3-9.153x2＋55.727x-95.819 （R2=0.953）. It was concluded that TMA-N in tissues could be used as a new estimator for late PMI. The method used in this study offered advantages such as accuracy, sensitivity, little samples required and wide PMI estimation.

Changes in ATP Levels in Rabbit Blood and Its Application for Estimation of the Postmortem Interval

Summary： Relationship between ATP changes of rabbit blood and postmortem interval （PMI） was studied. Twenty-four healthy rabbits were sacrificed and randomly divided into 3 groups with 8 rab- bits of each group. The bodies of three groups were placed in calorstat at temperature of 15℃, 25℃ and 35℃, respectively. The blood from the right ventricle was sampled through indwelling needle each 4 h until 72 h after death. ATP levels in the blood samples were measured by using ATP fluo- rescence rapid detection technique at different PMIs. Blood ATP levels slightly increased in the early stage after death and then constantly declined at all temperatures （15℃, 25℃, and 35℃）. Cubic polynomial regression equations with log[ATP] as dependent variable （y） and PMI as independent variable （x） at different temperatures and the optimal time period were established as followed： Under 15℃ and during 16-64 h after death, y=-3.027×10^-5x^3＋0.003x^2-0.096x-10.625 （Ra^2=0.992, P〈0.001）; under 25℃ and during 8-56 h after death, y=-2.921×10^-5x^3＋0.002x^2- 0.059x-11.186 （Ra^2=0.989, P〈0.001）; under 35℃ and during 4-36 h after death, y=-9.769×10^-5x^3＋ 0.005x^2 -0.117x-11.166 （Ra^2=0.991, P〈0.001）. The changes in ATP levels in blood collected from right ven- tricle of rabbit cadavers showed relatively stable and regular degradation within 72 h after death at different temperatures.

Image Analysis on Corneal Opacity:A Novel Method to Estimate Postmortem Interval in Rabbits

Corneal opacity is one of the most commonly used parameters for estimating postmortem interval （PMI）. This paper proposes a new method to study the relationship between changes of corneal opacity and PMI by processing and analyzing cornea images. Corneal regions were extracted from images of rabbits＇ eyes and described by color-based and texture-based features, which could represent the changes of cornea at different PMI. A KNN classifier was used to reveal the association of image features and PMI. The result of the classification showed that the new method was reliable and effective.

Inferring the Postmortem Interval of Rat Cadaver after Boiling Water Treatment Based on Microbial Community Succession

Background:In forensic investigations,accurate estimation of the postmortem interval(PMI)is an important task,but also an ongoing challenge.Especially in cases where the cadaver has been specially treated,for example,by boiling,the determination of PMI becomes extremely difficult.Previous studies have shown that the succession of the microbial community after decomposition of the cadaver can be used to infer PMI.However,the feasibility of determining the PMI of boiled cadavers has not yet been demonstrated.Aims and Objectives:The main objective of this study was to test whether we can infer PMI of boiled cadavers based on the succession of microbial communities.Materials and Methods:SD rats were killed by cervical dislocation.Subsequently,the rat cadavers were divided into the case(boiled cadavers)and control(unboiled cadavers)groups.Rectal samples were collected from the rats for 45 days and at nine time points.High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial community in the rectum.Results:The results showed that the composition and relative abundance of bacterial communities at the phylum level were significantly different between the case and control groups.The alpha diversity of the microbial community showed a decreasing trend with the decomposition process.Principal coordinate analysis showed that the case and control groups had obvious patterns along the succession of microbial communities.The rectal microbial communities showed a significant linear trend in the time course of decomposition.A random forest model was used to infer PMI.The goodness-of-fit(R2)of the model was 68.00%and 84.00%,and the mean absolute errors were 2.05 and 1.48 days within 45 days of decomposition for the case and control groups,respectively.Conclusions:Our results suggest that microbial community succession could be a potential method to infer PMI of boiled cadavers.

Research progress in the estimation of the postmortem interval by Chinese forensic scholars

The determination of time since death or the postmortem interval(PMI)is one of the most important and frequently asked questions in forensic medicine.Medicolegal scholars and forensic pathologists around the world have studied the estimation of PMI extensively in the past,and many novel methods and advanced technologies have now been applied in the field.For several centuries,Chinese forensic examiners have also worked on the estimation of the PMI,and there are a large number of excellent studies published in Chinese rather than in English,and these are not easily accessible or known internationally.Therefore we have conducted a review of relevant studies published by Chinese forensic scholars in the last few decades.The scope of this review is to provide a concise summary of the current progress in the estimation of PMI by Chinese forensic researchers using molecular biology,spectroscopic technology,entomological methods,energy changes,thanatochemistry and other methods.

Determination of Electrical Conductivity of Cadaver Skeletal Muscle: A Promising Method for the Estimation of Late Postmortem Interval

The electrical conductivity(EC)of extracted muscle fluid has been extensively used to evaluate meat freshness and shelf life in the field of food sanitation for decades.The opposite of freshness is the corruption that increases with time.Based on the freshness/corruption principle,we investigated the relationship between long postmortem intervals(PMIs)and EC in cadaver skeletal muscle.EC values of extracted fluid from rat muscles were measured at different PMIs for 10 days.The results indicate that there was a significant correlation between PMI and EC,and the data fit well to the cubic polynomial regression equation y=‑0.01x3+0.264x2‑13.657x+1769.148(R2=0.925).In addition,the EC of different dilutions of these muscle extracts showed strict quadratic correlation(R2=1)with the dilution ratios,suggesting that EC can be measured with very small quantities of muscle sample.Our study suggests that determination of the EC of cadaver skeletal muscle extracts may be a useful method for estimating long PMIs.

Determining the Electrical Conductivity of Rat Cadaveric Liver,Spleen,and Kidney to Estimate Early Postmortem Interval

Previous research has found that electrical conductivity(EC),an important index to predict meat freshness and shelflife,is very promising for estimating the late postmortem interval(PMI).However,whether it has potential use in the early PMI has not been fully studied yet.To test this possibility in the present study,EC of three internal organs of rat liver,spleen,and kidney were determined within 24 postmortem hows,and then,EC changes at different PMIs were carefully analyzed.The overall results showed that EC of liver and spleen increased significantly with PMI while EC of kidney had minor changes during the same period.Specifically,significant linear positive correlations between EC of liver and spleen and PMI were found and the coefficients of their regression functions were R^(2)=0.98 and R^(2)=0.95,respectively.It can be concluded that determination of EC in certain internal organs such as liver and spleen may be a potential tool in the early PMI estimation.However,more researches on its influencing factors are needed to facilitate its final use in practice.

Estimation of Postmortem Interval Using the Radiological Techniques,Computed Tomography:A Pilot Study

Estimation of postmortem interval(PMI)has been an important and difficult subject in the forensic study.It is a primary task of forensic work,and it can help guide the work in field investigation.With the development of computed tomography(CT)technology,CT imaging techniques are now being more frequently applied to the field of forensic medicine.This study used CT imaging techniques to observe area changes in different tissues and organs of rabbits after death and the changing pattern of the average CT values in the organs.The study analyzed the relationship between the CT values of different organs and PMI with the imaging software Max Viewer and obtained multiparameter nonlinear regression equation of the different organs,and the study provided an objective and accurate method and reference information for the estimation of PMI in the forensic medicine.In forensic science,PMI refers to the time interval between the discovery or inspection of corpse and the time of death.CT,magnetic resonance imaging,and other imaging techniques have become important means of clinical examinations over the years.Although some scholars in our country have used modem radiological techniques in various fields of forensic science,such as estimation of injury time,personal identification of bodies,analysis of the cause of death,determination of the causes of injury,and identification of the foreign substances of bodies,there are only a few studies on the estimation of time of death.We detected the process of subtle changes in adult rabbits after death,the shape and size of tissues and organs,and the relationship between adjacent organs in three-dimensional space in an efiort to develop new method for the estimation of PMI.The bodies of the dead rabbits were stored at 20℃ room temperature,sealed condition,and prevented exposure to flesh flies.The dead rabbits were randomly divided into comparison group and experimental group.The whole-body CT scans were performed on the experimental group of rabbits at different PMIs.NeuViz dual-slice spiral CT scanner(made by Neusoft Medical in China,2 mm×10 mm high-speed rare earth ceramic detector)is a 360°scan that could obtain two images,capable of providing a wide range of high-speed continuous spiral scan.Max Viewer(Version:1.0.0131,Neusoft,Shenyang,China)is a CT image viewing software developed by Neusoft Medical.The software can be used to view and process images in various common methods and to measure a number of parameters,such as length,area,angle,and CT values.Statistical analysis was performed using SPSS1 Statistics 19.0(SPSS,Inc.,Chicago,IL,USA).AP<0.05 was considered statistically significant.We obtained the binomial regression equation of the CT values and the related coefficient(R^(2)).In the future,we suggests that comprehensive analyses of various indicators of different organs could establish a diversified pattern to remedy the deficiencies and make the study of PMI estimation more scientific and enhance the operability.

Estimation of Postmortem Interval by Postmortem Myocardial Computed Tomography Value

Background:The estimation of postmortem interval(PMI)is one of the most important topics in forensic medicine research.We speculate that with an increased PMI,the computed tomography(CT)values of different tissues may show regular changes.Purpose:To use postmortem computed tomography(PMCT)to measure the myocardial CT value(unit:Hounsfield Unit,HU)of the heart to explore its pattern in postmortem change,and to discuss whether it can serve as a new parameter for PMI estimation.Methods:A total of 10 healthy adult New Zealand rabbits were selected and then put into a 20℃ incubator after being sacrificed.Within 0-156 h after death,CT scans were performed every 12 h to detect changes in the myocardial CT value of the heart over time.Regression analysis was used to determine the relationship between the myocardial CT value of the heart and PMI.At the same time,HE and Masson were used to stain the cardiac tissue sections detected by PMCT at Oh,48h and 156h,respectively.Results:During 0-156 h,the overall myocardial CT value showed a trend of first rising and then decreasing with the increase of PMI.The fitting regression equation was y=-2873.193+143.866x-1.728x2(x:myocardial CT value,unit:Hu;y:PMI,unit:h,R2=0.466,P<0.05).During 48-156 h,the overall myocardial CT value decreased gradually with the increase of PMI.The fitting regression equation was y=-93.038+18.700x-0.321x2(x:myocardial CT value,unit:Hu;y:PMI,unit:h,R2=0.963,P<0.001).The results of the morphological changes of the myocardial tissue structure after death showed that the myocardial cell structure was relatively complete at 0-48 hours after death;and the myocardial cell structure disappeared at 156 hours after death.Conclusions:Our results revealed evident postmortem changes in the myocardial CT value of the heart.Accordingly,measuring the myocardial CT value through PMCT shows promise for being used as a parameter for PMI estimation in forensic medicine and is worthy of further studies.The morphological changes of the myocardial tissue structure after death provide morphological basis for postmortem changes of tissue density,and further prove the reasons for the changes of CT value.

High-throughput sequencing to evaluate the effects of methamphetamine on the succession of the bacterial community to estimate the postmortem interval

In forensic medical examinations,estimating the postmortem interval(PMI)is an important factor.Methamphetamine(MA)is a synthetic stimulant that is commonly abused,and estimation of the PMI after MA abuse has become one of the main tasks in forensic investigation.Microorganisms play a vital role in carrion decomposition.Analysing the bacterial succession patterns can be used as a forensic tool to estimate the PMI.The present study aimed to analyse bacterial succession changes during the decomposition of MA to estimate the PMI.We analysed bacterial communities in rabbits treated with three different concentrations of MA(0,22.5,and 90 mg/kg)under the natural conditions of 20°C and 70%humidity by sequencing 16S rRNA gene amplicons using the Illumina MiSeq system.We obtained 2374209 high-quality sequences and 2937 operational taxonomic units(OTUs).The relative abundances of the bacterial communities varied markedly in response to different MA concentrations.Interestingly,in response to the different concentrations of MA,Bacteroidetes became disparate in the rectum in the late PMI.Increased numbers of bacterial taxa were identified in the rectum and buccal cavity samples,except at the highest concentration of MA in the rectum samples when PMI was 0-h,than were present in live rabbits.Meanwhile,the PMI correlated significantly with bacterial succession at different taxonomic levels.Our results suggested that bacterial community succession could be used as a“microbial clock”to estimate the PMI in cases of MA-related death;however,further study is required to gain a deeper understanding of this concept.

Variation in decomposition stages and carrion insect succession in a dry tropical climate and its effect on estimating postmortem interval

Insects have an important role in minimum postmortem interval(PMImin)estimation.An accurate PMImin estimation relies on a comprehensive study of the development and succes-sion of local carrion insects.No published research on carrion insect succession exists for tropical north Queensland.To address this,we aimed to obtain preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in Townsville and compare these with other regions of Australia and overseas.Adult insects were collected daily from three pig carcasses for 30d during summer and identified to fa-mily level.Observations of decomposition rate were made each day and progression through the stages of decomposition were recorded.Adult insects were identified to family and their presence/absence used as a proxy for arrival at/departure from the remains,respectively.These preliminary data highlight several interesting trends that may be inform-ative for forensic PMImin estimation.Decomposition was rapid:all carcasses were at the dry/remains stage by Day 5,which was substantially quicker than all other regions in the com-parison.Differences were also observed in the presence/absence of insect families and their arrival and departure times.Given the rapid progression through early decomposition,we argue that later-arriving coleopteran taxa may be more forensically informative in tropical Australia,in contrast with temperate regions where Diptera appear most useful.This research contributes preliminary observational data to understanding insect succession pat-terns in tropical Australia and demonstrates the critical need for comprehensive local succes-sion data for each climatic region of Australia to enable accurate PMImin estimation.These data will inform future research targeted at gaining a more comprehensive understanding of insect succession in the Australian tropics.

An Emergence of Dental Tissues in the Forensic Medicine for the Postmortem Interval Estimation:A Scoping Review

It is observed that there is not enough literature to support the usefulness of dental tissue in forensic medicine for the estimation of postmortem interval(PMI).Therefore,the authors of this article have systematically reviewed the available literature to understand the most used dental tissues,the methods employed to evaluate these tissues,and their reliability in PMI estimation.A MEDLINE-PubMed search engine was used to review the literature using the keywords“teeth,”“dental tissues,”“post‑mortem interval,”and“forensic dentistry”from 2001 until 2020.The most used tissue was dental pulp,but other cellular‑.and molecular‑level dental tissues such as enamel,dentin,cementum,periodontal ligament,odontoblast,and mitochondrial and nuclear DNA and RNA were also used.Based on this observation,the authors have provided the categorical classification to evaluate these dental tissues into morphological,histopathological,and molecular‑level examination to understand the reliability of these dental tissues.The molecular examinations to evaluate these tissues were widely employed and emerging in the estimation of PMI as this method could provide a reliable outcome when compared with others.Since the literature reviewed was not enough to answer our questions,however,to conclude,the dental pulp is the widely used tissue so far.Further,more studies are required to authenticate the role of other dental tissues to predict the PMI at an early stage with cost‑effective and most convenient techniques.

Estimation of the Postmortem Interval Using Chromatographic Fingerprints of Volatile Organic Compounds from Muscle

Estimation of the postmortem interval(PMI)is a crucial task in the field of forensic pathology and has unfortunately not been properly resolved.In this study,we analyzed volatile organic compounds(VOCs)in rat muscle samples collected at different PMIs and studied the feasibility of muscle VOC fingerprinting as a new method for PMI estimation.In total,110 rats were sacrificed and stored at a constant temperature(25℃).Rat skeletal muscle samples were collected at 0-10‑day postmortem,and then the VOCs were determined using a method of headspace solid‑phase microextraction coupled with gas chromatography‑mass spectrometry.The correlations between the VOCs(species and quantities)and PMIs were carefully analyzed and standard muscle VOC fingerprints at 25℃were established for different PMIs.To further test the accuracy of muscle VOC fingerprinting as a method for PMI estimation,ten additional rats with known PMIs were studied.We identified 15 kinds of VOCs and the number of VOC species increased with the PMI.The total peak areas of the VOCs increased significantly with the postmortem day(adjusted R^(2)=0.96-0.97).The mean error of the VOC fingerprinting for PMI estimation was 0.5 days and the mean relative error was 8.33%.We concluded that muscle VOC fingerprinting combining the use of VOC species and peak areas is accurate and effective and could be used as an alternative approach for PMI estimation in forensic practice.Although the preliminary results are encouraging,further studies in human cadavers under real case conditions are needed.

Vitreous Humor:A Review of Biochemical Constituents in Postmortem Interval Estimation

Postmortem changes in the biochemical constituents of the vitreous humor have been widely used to estimate the postmortem interval(PMI)over the past several decades.However,few reviews have summarized the relationship between the postmortem vitreous biochemical constituents and time of death.Herein,the relationship between PMI and single biochemical components,including vitreous potassium,hypoxanthine,and amino acids,as well as comparisons of each statistical parameter in the formula,is summarized.We also discuss other compounds such as urea and uric acid,which have no direct relationship with PMI.Utility of multiple constituent simultaneous analysis for estimating PMI is being increasingly investigated.The promising idea of using simultaneous analysis of multiple constituents to determine PMI is proposed as a future research direction.

Estimation of the Postmortem Interval by Measuring Blood Oxidation‑reduction Potential Values

Accurate estimation of the postmortem interval(PMI)is an important task in forensic practice.In the last half-century,the use of postmortem biochemistry has become an important ancillary method in determining the time of death.The present study was carried out to determine the correlation between blood oxidation-reduction potential(ORP)values and PMIs,and to develop a three-dimensional surface equation to estimate the PMI under various temperature conditions.A total of 48 rabbits were placed into six groups and sacrificed by air embolism.Blood was obtained from the right ventricle of each rabbit,and specimens were stored at 10℃,15℃,20℃,25℃,30℃,and 35℃.At different PMIs(once every 4 h),the blood ORP values were measured using a PB-21 electrochemical analyzer.Statistical analysis and curve fitting of the data yielded cubic polynomial regression equations and a surface equation at different temperatures.Result:The results showed that there was a strong positive correlation between the blood ORP values at different temperatures and the PMI.This study provides another example of using a three-dimensional surface equation as a tool to estimate the PMI at various temperature conditions.

Estimation of Postmortem Interval Using Attenuated Total Reflectance:Fourier Transform Infrared Spectroscopy in Adipose Tissues

Estimation of postmortem interval(PMI)is vitally important in forensic investigations.Although many studies have examined the chemical changes of various tissues over time,no reports using spectroscopic methods in adipose tissue are available.In this study,attenuated total reflectance-Fourier transform infrared(ATR-FTIR)spectroscopy was utilized to collect comprehensive biochemical information from human adipose tissues in vitro at different times.Thereafter,mice were used as samples for in vivo experiments for more detailed studies on PMI.Then,partial least squares(PLS)model for PMI estimation was established based on the acquired spectral dataset of mouse samples.The spectral variable associated with C=O arising from lipids and free fatty acids was most susceptible to PMI.Moreover,the PLS model appeared to achieve a satisfactory prediction with a root mean square error of cross‑validation of 1.78 days,and the reliability of the model was determined in an external validation set with a root mean square error of prediction of 1.87 days.The study shows the possibility of application of ATR-FTIR methods in PMI estimation using adipose tissue.

DNA degradation within mouse brain and dental pulp cells 72 hours postmortem

In this study, we sought to elucidate the process of DNA degradation in brain and dental pulp cells of mice, within postmortem 0-72 hours, by using the single cell gel electrophoresis assay and professional comet image analysis and processing techniques. The frequency of comet-like cells, the percentage of tail DNA, tail length, tail moment, Olive moment and tail area increased in tandem with increasing postmortem interval. In contrast, the head radius, the percentage of head DNA and head area showed a decreasing trend. Linear regression analysis revealed a high correlation between these parameters and the postmortem interval. The findings suggest that the single cell gel electrophoresis assay is a quick and sensitive method to detect DNA degradation in brain and dental pulp cells, providing an objective and accurate new way to estimate postmortem interval.

根据人补体第三成分裂解率推断死亡时间

本文应用交叉免疫电泳方法研究离体全血和尸血补体第三成分（C3）的裂解率与死后时间的关系。将离体全血孵育于不同温度与时间，观察到温度越高、时间越长，C3裂解速度越快。死亡时间与尸血C3裂解率呈正相关关系，其推断死亡时间95％可信区间为死亡时间±12h。若同时考虑尸体直肠温度，推断死亡时间95％可信区间为死亡时间±6h。说明，根据C3裂解率可较准确地推断死亡时间。

Correlative Analysis on the Relationship between PMI and DNA Degradation of Cell Nucleus in Human Different Tissues

Summary： To determining the postmortem interval （PMI） through quantitative analysis of the DNA degradation of cell nucleus in human brain and spleen by using image analysis technique （IAT）. The brain and spleen tissues from 32 cadavers with known PMI were collected, subjected to cell smear every 1 h within the first 5-36 h after death, stained by Feulgen-Van＇s staining, Three indices reflecting DNA in brain cells （astrocytes） and splenic lymphocytes, including integral optical density （IOD）, average optical density （AOD）, average gray （AG） were measured by employing the mage analysis instrument. The results showed that IOD and AOD declined and AG increased with the prolongation of dead time within 5 36 h. A correlation between the PMI and gray parameters （IOD,AOD and AG） was identified and the corresponding regression equation was obtained. The parameters （IOD, AOD and AG） were proved to be effective quantitative indicators for accurate estimation of PMI within 5-36 h after death.