A statistical analysis of successional patterns in carrion-arthropod assemblages: implications for forensic entomology and determination of the postmortem interval.

A set of statistical protocols is proposed for analyzing carrion-arthropod succession in forensic entomology investigations. A total of 23 carrion-arthropod data sets from temperate, tropical, desert, and coastal environments were assembled in a standard format and analyzed using randomization tests and methods derived from quantitative community ecology. The data were analyzed in three ways. First, patterns of arthropod visitation on nonhuman carcasses were analyzed in each of the 23 cases. Analysis revealed two groups of taxa: those that persist on the carcass over a single time interval (= nonreoccurring taxa, 80% of the taxa) and those that appear, leave, and reappear over time on the carcass (= reoccurring taxa, 20%). Reoccurring taxa, which can confound estimation of the postmortem interval (PMI), were found in 6 classes, 12 orders, and 29 taxonomic families of arthropods, including some forensically important taxa (for example, calliphorids, sarcophagids, histerids). The recognition that reoccurring taxa exist and that they are found in forensically important groups is an early step toward factoring in their potential importance in future entomological investigations. Second, temporal changes in the taxonomic composition of the carrion-arthropod community were studied by quantifying the degree of taxonomic similarity between pairwise combinations of time-specific samples of the succession. In 13 of the 18 illustrated cases, the midsuccessional samples, owing to their higher species richness, were taxonomically more similar to all other pairwise samples, on the average, than early and late successional samples which were poorer in species. Variability in taxonomic composition is the norm for most periods of the succession; however, in 17 of the 23 cases, some successional periods (particularly endpoint samples) revealed no changes in arthropod species composition (= matching sample-pairs). When applied to medicolegal cases, it is suggested that data sets with large fractions of matching sample-pairs should produce wider-ranging PMI estimates, on the average, than data sets with smaller fractions of matching sample-pairs. Third, Monte Carlo simulation was applied to each of the 23 assemblages to test specific hypotheses about community-wide patterns of arthropod visitation times on nonhuman carcasses. Simulation results revealed that arthropod residence times in the majority of cases (13 or 56%) followed a "clumped" succession model, whereas, the remaining 10 cases (44%) showed a more "uniform" spacing pattern of residence times on carcasses. Comparison of species accumulation curves for observed and simulated data further revealed that among the 13 "clumped" cases, most (9 or 69%) followed a "clumped, early" model (rather than "clumped, midterm" or "clumped, late" models).(ABSTRACT TRUNCATED AT 400 WORDS)     

Factors affecting decomposition and Diptera colonization.

Understanding the process of corpse decomposition is basic to establishing the postmortem interval (PMI) in any death investigation even using insect evidence. The sequence of postmortem changes in soft tissues usually gives an idea of how long an individual has been dead. However, modification of the decomposition process can considerably alter the estimate of the time of death. A body after death is sometimes subject to depredation by various types of animals among which insects can have a predominant role in the breakdown of the corpse thus, accelerating the decomposition rate. The interference of the insect community in the decomposition process has been investigated by several experimental studies using animal models and very few contributions directly on cadavers. Several of the most frequent factors affecting PMI estimates such as temperature, burial depth and access of the body to insects are fully reviewed. On account of their activity and world wide distribution, Diptera are the insects of greatest forensic interest. The knowledge of factors inhibiting or favouring colonization and Diptera development is a necessary pre-requisite for estimating the PMI using entomological data.     

Post-feeding larval behaviour in the blowfly, Calliphora vicina: effects on post-mortem interval estimates

Using the rate of development of blowflies colonising a corpse, accumulated degree hours (ADH), or days (ADD), is an established method used by forensic entomologists to estimate the post-mortem interval (PMI). Derived from laboratory experiments, their application to field situations needs care. This study examines the effect of the post-feeding larval dispersal time on the ADH and therefore the PMI estimate. Post-feeding dispersal in blowfly larvae is typically very short in the laboratory but may extend for hours or days in the field, whilst the larvae try to find a suitable pupariation site. Increases in total ADH (to adult eclosion), due to time spent dispersing, are not simply equal to the dispersal time. The pupal period is increased by approximately 2 times the length of the dispersal period. In practice, this can introduce over-estimation errors in the PMI estimate of between 1 and 2 days if the total ADH calculations do not consider the possibility of an extended larval dispersal period.     

Biolog-Eco法检测尸体微生物群落的代谢功能变化

目的检测死后不同时间尸体上微生物群落功能多样性的变化并评估其在死亡时间推断中的应用价值。方法采用Biolog-Eco微平板对野外实验中来自于死后0~240 h猪和人尸体的肛门拭子进行微生物群体的培养,监测其引起的光密度值变化,结合法医病理学及蝇类演替,观察自然腐败尸体微生物群落的代谢特性及其变化。结果微生物代谢功能多样性与蛆虫数量变化呈负相关关系。冷冻在0 h对每孔颜色平均变化率影响最大,48 h后基本消失,192 h后尸体微生物群落多样性相对不稳定。主成分分析将31种碳源综合为5个主成分(累积贡献率90%)。碳源tsquare分析显示,N-乙酰-D-葡萄糖氨和L-丝氨酸是推断人和猪样品PMI(0~240 h)的优势碳源。结论 Biolog-Eco法能表现死亡后0~240 h尸体上微生物群落对部分碳源利用的代谢差异,有望为死亡时间推断提供新依据。     

Flies eggs: a new method for the estimation of short-term post-mortem interval?

Three modeling methods were used to estimate the time for laying of Lucilia sericata eggs (Diptera, Calliphoridae) after measurements of hatching times at several constant temperatures. These models were tested first under controlled conditions with 19 profiles of fluctuating temperatures, and on the other hand under field conditions on mice. All three models were allowed to determine the time of laying within a period of about 2h, and consequently this determines the time of death, as this species lays immediately after death under favorable conditions. Rearing of eggs sampled on a corpse may therefore contribute to the determination of a short post-mortem interval (PMI).     

A Forensic Entomological Analysis Can Yield an Estimate of Postmortem Interval,and Not Just a Minimum Postmortem Interval: An Explanation and Illustration Using a Case

Several authors claimed that a forensic entomological analysis can never be interpreted as the postmortem interval (PMI), but that it can be interpreted as a minimum postmortem interval (PMI_min), or dead for not less than X amount of time. Because in most instances, a PMI estimate should be a range, that is, the set of values from maximum postmortem interval (PMI_max) to PMI_min, objections to PMI estimation are objections to PMI_max estimation. The arguments for this position did not address the substantial body of literature describing estimation of both PMI_max and PMI_min using insect succession analysis. This report reviews the scientific issues and presents a recent homicide investigation in which several forensic entomology experts used the absence of carrion insects on the corpse, a kind of succession analysis, to estimate PMI or PMI_max.     

Estimation of postmortem interval in real cases based on experimentally obtained entomological evidence

Using the entomological evidence obtained in several forensic cases analyzed in our laboratory for comparison, we evaluated the results of an experimental study carried out in a semiurban setting to determine the structure of the sarcosaprophagous fauna from a Mediterranean region of SE Spain. In all, 18 orders of arthropods were collected. The summarized experimental results refer to the most important taxa for estimating the postmortem interval. Thus, the seasonal character of certain Diptera species, such as Phaenicia sericata, Calliphora vicina, Chrysomya albiceps and Musca domestica, is cited. Among the Coleoptera, the role of Dermestidae as a necrophagous species, and of Staphylinidae and Histeridae as necrophilous, or Cleridae, Tenebrionidae and Nitidulidae as omnivorous, as well as their appearance on the corpse, is described. Among the Hymenoptera, Formicidae were as the most abundant group, acting as omnivores and not apparently related to any particular decomposition stage. The real cases are discussed using data from the literature and the experimentally obtained results. In every case, the most relevant factors for estimating PMI are briefly discussed, mentioning, when possible, the relation with the experimental results. We confirm the significance of the experimental results because they seem to be applicable to actual forensic cases, the details of which enlarge our very little knowledge on the subject in the Iberian Peninsula. The importance of regional faunistic studies of the sarcosaprophagous arthropod community, the results of which may be applied to forensic practice, is mentioned.     

The selection of endogenous genes in human postmortem tissues

Precisely determining the postmortem interval (PMI), which is crucial to criminal and forensic cases, is a research in which quantitative RT-PCR (also known as qRT-PCR or real-time RT-PCR) has been used to analyse gene expression levels and data normalisation should be required to eliminate the differences among the samples. Therefore, it is quite necessary to find stable molecular biological markers in PMI determination research. In this study, we compared nine commonly used endogenous markers (containing ACTB, GAPDH, B2M, U6, 18S rRNA, hsa-mir-1, hsa-mir-9, hsa-mir-194-1 and hsa-mir-203) in the 109 human tissue samples obtained from autopsy at the aim of finding stable markers in human tissues with consideration of the impact of parameters (PMI and cause of death). After RNA was extracted from four tissues (heart, brain, kidney, skin), the Ct values of nine endogenous markers were obtained by qRT-PCR and assessed by geNorm software. The results showed that U6, GAPDH and 18S rRNA were the suitable markers in our set of samples in various corpse conditions, that B2M and ACTB were reliable internal controls in heart tissue only, and that microRNAs had such high M values that they should not be chosen for endogenous control genes.     

Improved estimation of postmortem interval based on differential behaviour of vitreous potassium and hypoxantine in death by hanging

Many formulae are available to estimate the relation between the potassium ([K+]) and hypoxantine ([Hx]) concentration in the vitreous humour and the postmortem interval (PMI). Typically these have been based on a correlation test and linear regression using the postmortal interval as the independent variable and [K+] or [Hx] as the dependent variable in order to estimate the confidence interval. However, a recent study has shown that a more precise measurement of PMI can be obtained if [K+] is used as the independent variable. The regression lines obtained from the most recent deceased subjects with forensic relevance received for autopsy in the Institute of Legal Medicine are [K+] = 5.589 + 0.174PMI and [Hx] = 26.459 + 3.017PMI, by changing the variables, we obtain PMI=3.967[K+] - 19.186 (R2 = 0.688, P < 0.001) and PMI = 0.172 [Hx] + 0.170 (R2 = 0.518, P < 0.001). In this paper we propose the cause of death as an extra factor which modifies the relationship and gives even greater precision in estimating PMI. In cases of death by hanging the results are considerably improved with [K+] = 5.224 + 0.225PMI and [Hx] = 15.161+4.957PMI, respectively, and consequently, PMI = 3.631[K+] - 17.334 (R2 = 0.818, P< 0.001) and PMI = 0.153[Hx] - 0.368 (R2 = 0.757, P < 0.001): the slope is less and the precision is obviously enhanced.     

Experimental validation of forensic evidence: a study of the decomposition of buried pigs in a heavy clay soil.

In a murder investigation, where the victim had been strangled and buried in a shallow grave, there were discrepancies between the post mortem interval (PMI) as estimated from entomological studies and estimations determined from other evidence. This inconsistency provided the impetus for examining the decay process using pig carcasses as analogues for the human cadaver. The pigs were buried in the immediate vicinity of the original burial site in December 1996, which was the month when the victim was purported to have been interred in the previous year. The buried pigs were then monitored for 5 months which, based on the evidence other than the entomological, was the period over which the corpse was thought to have lain in the ground. The pig corpses were disturbed by scavengers in mid April: this was the same time that the human corpse was discovered in the previous year by scavengers. Insects played no role in the decomposition process until the pig carcasses had been exposed by animals. Blowflies, notably Calliphora vomitoria, were attracted to the exposed tissues and laid eggs from which larvae developed. Calliphora vomitoria is a species often used to estimate PMI. This investigation has shown that soil conditions and low seasonal temperatures had preserved the pig carcasses for longer than might be expected. Using the blowfly larvae to estimate PMI would have produced erroneous results had not the burial environment and exhumation history been investigated.     

组织细胞DNA含量变化与死亡时间的相关性研究

目的研究人死后各器官组织细胞DNA降解变化规律,探讨其在推断死亡时间方面的应用价值,为推断死亡时间提供科学依据。方法已知死亡时间人尸体的心、肝、脾、肾,按死后6,12,24和48h四个时间段取材,制成石蜡切片,经Feulgen染色并图像分析;同时将各器官组织按不同时间段取材,制成单细胞悬液,应用流式细胞仪检测DNA含量。结果(1)人体各器官组织细胞经FCM检测,脾细胞核DNA随死亡时间延长呈现较好的下降梯度,而心、肝、肾,则由于自溶较快,死后6h细胞核DNA含量已很低,其变化情况与死亡时间的相关性不佳;(2)各器官组织细胞核DNA染色强度指数均随死亡时间的延长而下降。结论(1)机体死亡后,各器官组织细胞核DNA含量随死亡时间延长逐渐减少,具有一定的变化规律,其中以脾细胞核DNA含量的变化趋势与死亡时间最具相关性,肝、肾次之,而心最差;(2)方法学分析,图像分析技术(IAT)与流式细胞术(FCM)两种检测方法在研究组织细胞DNA含量变化方面,各有利弊,可互补不足。     

Methods used for the killing and preservation of blowfly larvae, and their effect on post-mortem larval length

A record of the length of the largest larvae collected from a corpse can be used to estimate the age of the oldest larvae present and, therefore, give an estimate of minimum time since death. Consequently, factors that affect post-mortem larval length will impact on any estimate of PMI based on it. Methods used to kill and preserve larvae are known to affect post-mortem length. This study looks at the effects of different preservatives, and variations in the protocol used for killing larvae by immersion in a hot water bath = [hot water killed; HWK], on the length of dead larvae of two common blowfly species. Post-feeding third instar Calliphora vomitoria and Lucilia sericata larvae were either HWK in boiling water and then placed in 80% ethanol or 10% formaldehyde solution, or placed live into the preservatives. For both species, choice of preservative and method of killing significantly affected post-mortem length. There were significant interspecific differences in their response to identical methods of killing and preservation. Additional experiments were carried out where C. vomitoria larvae were HWK in water at 80 and 100 degrees C for 1, 30, 60 and 90 s duration. Both temperature and duration significantly affected post-mortem length. Maximum length was attained after at least 60 s immersion. The amount of post-mortem decomposition that occurred after the larvae were placed in preservative could be greatly reduced by increasing the duration of immersion and/or increasing the water temperature. For the HWK larvae, it was possible to record their length immediately after death and before they had been placed in preservative. This data revealed that where 80% ethanol was used as a preservative the larvae expanded in the preservative. The timing of this expansion was investigated with a sample of C. vomitoria, HWK at 100 degrees C for 30 s and recording post-mortem length immediately after death and again after 3, 6, 9, 12, 24, 27, 30 and 33 h storage in 80% ethanol. Maximum length was recorded after 12 h storage and the rate of expansion was highest during the first 3 h in this preservative. After long-term storage (290 days), larvae killed and preserved in the same way were on average 0.7% longer than immediately after death and 0.6% (0.11 mm) smaller than when last measured (after 28 days storage).     

Blind validation of postmortem interval estimates using developmental rates of blow flies

This study evaluated the use of different degree day (DD) models, developmental thresholds and developmental data sources for estimating postmortem interval (PMI) based on developmental rates of blow flies (Diptera: Calliphoridae). Blow fly evidence was collected from three mock crime scenes and sent for blind analysis of PMI. PMI estimates were calculated using averaging, single sine, double sine, single triangle, and double triangle models of DD calculation with minimum developmental thresholds of 0, 6 and 10 °C and Anderson's, Byrd and Allen's, Greenberg's, Nishida's and Kamal's developmental data sets. These estimates were compared with the actual PMI to determine which combination of DD model, developmental threshold and developmental data provided the most accurate estimate. For all three cases, at least one method gave a PMI estimation that was within 1 day of the actual date of death. There was no variability between PMI estimated using the five models of DD calculation. Overall, as lower developmental threshold increased, the PMI interval estimates increased. Depending on the developmental data set used, increasing the lower developmental threshold caused some overestimations of the date of oviposition, with estimates given that were prior to the actual death. Future PMI estimates should include error ranges, so that overestimation of PMI is avoided.     

A retrospective comparative study to evaluate the reliability of post-mortem interval sources in UK and US medico-legal death investigations

Post-mortem interval (PMI) information sources may be subject to varying degrees of reliability that could impact the level of confidence associated with PMI estimations in forensic taphonomy research and in the practice of medico-legal death investigation. This study aimed to assess the reliability of PMI information sources in a retrospective comparative analysis of 1813 cases of decomposition from the Allegheny County Office of the Medical Examiner in Pittsburgh, US (n = 1714), and the Crime Scene Investigation department at Southwest Forensics in the UK (n = 99). PMI information sources were subjected to a two-stage evaluation using an adapted version of the 3x5 aspects of the UK police National Intelligence Model (NIM) to determine the confidence level associated with each source. Normal distribution plots were created to show the distribution frequency of the dependent variables (decomposition stage and source evaluation) by the independent variable of PMI. The manner, location, and season of death were recorded to ascertain if these variables influenced the reliability of the PMI. A confidence matrix was then created to assess the overall reliability and provenance of each PMI information source. Reliable PMI sources (including forensic specialists, missing persons reports, and digital evidence) were used across extensive PMI ranges (1 to 2920 days in the US, and 1 to 240 days in the UK) but conferred a low incidence of use with forensic specialists providing a PMI estimation in only 35% of all homicide cases. Medium confidence PMI sources (e.g., last known social contact) accounted for the majority of UK (54%, n = 54) and US (82%, n = 1413) cases and were associated with shorter PMIs and natural causes of death. Low confidence PMI sources represented the lowest frequencies of UK and US cases and exclusively comprised PMI information from scene evidence. In 96% of all cases, only one PMI source was reported, meaning PMI source corroboration was overall very low (4%). This research has important application for studies using police reports of PMI information to validate PMI estimation models, and in the practice of medico-legal death investigation where it is recommended that i) the identified reliable PMI sources are sought ii) untested or unreliable PMI sources are substantiated with corroborating PMI information, iii) all PMI sources are reported with an associated degree of confidence that encapsulates the uncertainty of the originating source.     

Comparison of chemical methods for determining postmortem interval

Accurate determination of postmortem interval (PMI) is a problem for the forensic thanatologist, especially in unwitnessed deaths. A number of objective chemical methods for determining PMI have been developed, the most widely used being accumulation of potassium in the vitreous humor. The authors previously have reported a chemical method for determining PMI from the predictable accumulation or clearance of the dopaminergic metabolite 3-methoxytyramine (3-MT) in the putamen of the brain. They have extended their previous study to compare directly the accuracy of determining PMI from the level of 3-MT in putamen with the level of potassium in vitreous humor. The data indicate that 3-MT is at least as accurate as, if not more accurate than, potassium accumulation in vitreous humor, although 3-MT levels can be affected by the cause of death and drugs present at the time of death. Nevertheless, determination of both the 3-MT and potassium levels can afford the most accurate method of determining PMI; preliminary nomograms for determining PMI from both variables are presented.     

人脾脏细胞核DNA含量与死亡时间关系的图像分析

目的研究人脾脏细胞DNA含量改变与死亡时间的关系。方法选取36例已知死亡时间的人体脾脏,在死后5～36h内逐时进行细胞学涂片、Feulgen Vans染色观察,并用图像分析系统,测定脾脏细胞核积分光密度、平均灰度等灰度参数,所得数据用SPSS软件分析。结果在5～36h内平均光密度、积分光密度、平均灰度均值均与死亡时间显著性相关,并得出对应的回归方程,其中平均光密度、积分光密度随死亡时间的延长而逐渐减小、平均灰度逐渐增大。结论人脾脏细胞核DNA含量改变呈现一定规律,与死亡时间明显相关。     

人体死后肝细胞DNA含量与死亡时间关系的研究

目的研究人体死后肝脏细胞DNA含量变化与死亡时间的关系及影响因素。方法选取46例已知死亡时间的人体肝脏,根据离体肝脏所处的环境温度分为12—19％（A组）和20—27％（B组）两组,每组23例。在死后24～72h内每隔4h穿刺取肝组织1次,制成细胞悬液,经RNA酶消化,PI染色后,用流式细胞仪测定被检测细胞中含不完整DNA的细胞数所占百分比,所得数据经Exp032V1．2软件计算N值。结果死后24～72h肝细胞N平均值,A组从10．91％增至49．72％,B组从16．22％增至69．63％。两组N平均值随死亡时间的延长均逐渐增高,与死亡时间有相关性,A组r值为：0．598,B组r值为0．77357。并且建立了不同环境温度对应的回归方程。结论在不同环境温度下,死后24～72h内人体肝脏细胞DNA降解均随死亡时间的延长和环境温度的升高而逐渐加快,相关数据可望为死亡时间推断提供一定参考依据。     

The effects of larval crowding and food type on the size and development of the blowfly, Calliphora vomitoria

The use of entomological evidence in the estimation of the post mortem interval (PMI) often depends on the size and developmental stage of blowfly larvae collected from a corpse. Therefore, factors which can have an effect on the larval size and growth rate can have implications for reliable PMI determinations. This study explores the competitive effects of larval overcrowding on Calliphora vomitoria reared on three different pig tissues--liver, brain and muscle. The competitive feeding environment within the more crowded larval cultures resulted in increased development rates and the production of undersized larvae and adults. Variation in the extent of these effects was observed on each of the three body tissues, highlighting the importance of documenting the positions from which entomological evidence is recovered from a corpse.     

家兔眼玻璃体液21例元素含量PMI关系的研究

目的寻找一种精确推定PMI的方法。方法应用电感耦合等离子体质谱(ICP-MS),系统研究了家兔死后96h内眼玻璃体液21种元素含量与PMI的相关性。结果眼玻璃体液Co、Mo、Cd、Sb、I、Pb、Bi、Li共8种元素与PMI无关。K、Mg、Fe、P、Na、Al、Ca、Ti、Ni、Cu、Zn、Sr、Ba共13种元素含量与PMI有关。结论眼玻璃体液K、Mg、Fe、P、Na、Al、Ca、Ti、Ni、Cu、Zn、Sr、Ba共13种元素与PMI有关。为研究人尸体眼玻璃体液元素与PMI的关系奠定了选择指标的基础。