Marcel A. Verhoff

De novo transcriptome analysis and highly sensitive digital gene expression profiling of Calliphora vicina (Diptera: Calliphoridae) pupae using MACE (Massive Analysis of cDNA Ends)

Determining a post-mortem interval using the weight or length of blow fly larvae to calculate the insects age is well established. However, to date, there are only a handful studies dealing with age estimation of blow fly pupae, in which weight or length cannot be used as a relevant parameter. The analysis of genetic markers, which indicate a certain developmental stage, can extend the period for a successful post-mortem interval determination. In order to break new ground in the field of age determination of forensic relevant blow flies, we performed a de novo transcriptome analysis of Calliphora vicina pupae at 15 different developmental stages. Obtained data serve as base to establish molecular age determination techniques. We used a new, deeper, and more cost-effective digital gene expression profiling method called MACE (Massive Analysis of cDNA Ends). We generated 15 libraries out of 15 developmental stages, with 3-8 million reads per library. In total, 53,539 distinct transcripts were detected, and 7548 were annotated to known insect genes. The analysis provides high-resolution gene expression profiles of all covered transcripts, which were used to identify differentially expressed genetic markers as candidates for a molecular age estimation of C. vicina pupae. Moreover, the analysis allows insights into gene activity of pupal development and the relationship between different genes interesting for insect development in general.

Establishment of developmental charts for the larvae of the blow fly Calliphora vicina using quantile regression.

Developmental data of necrophagous blow fly species can be used to estimate a minimum postmortem interval (PMImin) in death investigations by estimating the age of larvae sampled from the cadaver. The most important parameter used in this age estimation is the increase in larval length during growth. Larval length can be compared to species-specific reference data to get an age estimate for the larval specimen. The exploration of this type of data and the use of an appropriate statistical method are the major challenges in evaluating forensic entomological data sets. In Europe, Calliphora vicina is one of the most dominant species with forensic entomological relevance. Despite its frequency and importance, there are currently no published developmental studies for German populations of this blow fly that can be referenced for larval age estimations. This is regrettable because the geographical origin of different populations may lead to phenotypic plasticity in the same species and population-specific growth patterns that differ from published data sets. To address this shortcoming, the objective of the present research was to generate growth data for C. vicina which can be used for age estimation in German casework. We present, for the first time, local developmental data for the larval stages of German C. vicina, reared at three constant temperatures (15, 20 and 25°C) and compare the results with published studies on C. vicina development from elsewhere. To analyse the development we chose a quantile mixed effects model because of its robustness and insensitivity towards outliers. Quantile regression was developed as an extension of the linear model to estimate rates of change in all parts of the distribution of a response variable and to discover more useful predictive relationships between variables. By applying a linear quantile mixed effect model, we estimated the 0.1 and 0.9 quantile functions of the larval age for each temperature. Graphically, these quantile functions act like reference bands and therefore, plots for larval stages 1-3 can be used to estimate the age of a larva. Nevertheless, the post-feeding stage still remains difficult to describe with linear models and need to be further investigated. A comparison of the results from this present study with the currently applied methods for age estimation of C. vicina still demonstrates the importance of collecting growth rates for local blow fly populations and of standardising experimental designs and statistical analyses.

High-resolution flat-panel volumetric CT images show no correlation between human age and sagittal suture obliteration—Independent of sex

This study investigated whether digital, high-resolution CT-images of the internal human sagittal suture structure include information that enables a novel method of age at death (aad) determination. To accomplish this, coronal, flat-panel-based volumetric computed tomography (eXplore Locus Ultra scanner) images were automatically analyzed by a software implementation of an algorithm that determines user independent whether a suture is open or closed. 29,205 images of the local vicinity of the sagittal suture of 164 males and 85 females of European descent were investigated separately for both sexes. We used conditional probabilities and a chi(2)-test to investigate whether there is a correlation between aad and suture obliteration or not. The computer-aided analysis enables us to handle huge volumes of data that could not otherwise be analyzed within a reasonable time frame. The implemented algorithm ensured a strongly reproducible, reliable, accurate, and fast differentiation between closed and open sutures. The evaluation of various statistical parameters suggests that there is no reason to assume a correlation between age and suture closure--with equal findings for both sexes. Therefore, we conclude that determination of aad based on the evaluation of sagittal suture obliteration is not possible. This agrees well - thus unsatisfactorily - with the recent literature.

Aging adult skull remains through radiological density estimates: A comparison of different computed tomography systems and the use of computer simulations to judge the accuracy of results

INTRODUCTION The objective of this article was to explore age-at-death estimates in forensic medicine, which were methodically based on age-dependent, radiologically defined bone-density (HC) decay and which were investigated with a standard clinical computed tomography (CT) system. Such density decay was formerly discovered with a high-resolution flat-panel CT in the skulls of adult females. The development of a standard CT methodology for age estimations--with thousands of installations--would have the advantage of being applicable everywhere, whereas only few flat-panel prototype CT systems are in use worldwide. METHODS A Multi-Slice CT scanner (MSCT) was used to obtain 22,773 images from 173 European human skulls (89 male, 84 female), taken from a population of patients from the Department of Neuroradiology at the University Hospital Giessen and Marburg during 2010 and 2011. An automated image analysis was carried out to evaluate HC of all images. The age dependence of HC was studied by correlation analysis. The prediction accuracy of age-at-death estimates was calculated. Computer simulations were carried out to explore the influence of noise on the accuracy of age predictions. RESULTS Human skull HC values strongly scatter as a function of age for both sexes. Adult male skull bone-density remains constant during lifetime. Adult female HC decays during lifetime, as indicated by a correlation coefficient (CC) of -0.53. Prediction errors for age-at-death estimates for both of the used scanners are in the range of ±18 years at a 75% confidence interval (CI). Computer simulations indicate that this is the best that can be expected for such noisy data. CONCLUSIONS Our results indicate that HC-decay is indeed present in adult females and that it can be demonstrated both by standard and by high-resolution CT methods, applied to different subject groups of an identical population. The weak correlation between HC and age found by both CT methods only enables a method to estimate age-at-death with limited practical relevance since the errors of the estimates are large. Computer simulations clearly indicate that data with less noise and CCs in the order of -0.97 or less would be necessary to enable age-at-death estimates with an accuracy of ±5 years at a 75% CI.

Investigative strategy for the forensic detection of sperm traces

In a retrospective study, the results from 786 samples of alleged sexual assaults during a 5-year period were evaluated. Of the samples, 758 were from female victims and 28 were from male victims. The material examined during this 5-year period consisted of 561 cotton swabs with swabs taken from the genitals, mouth, anus, or skin surface. In addition, textile products were examined 191 times, paper products 23 times, and other evidentiary materials 11 times. The acid phosphatase (acP) test was performed as a preliminary test for all samples, followed by microscopy after Baecchi staining. DNA analysis was performed on 74 samples following individual court orders. The retrospectively evaluated results from this period indicate that additional tests for the detection of sperm on textiles and paper products are dispensable after a negative acP test. This is different for genital swabs, since sperm could be found microscopically in 3% of cases with a negative acP test, and DNA analysis was also successful. However, an individual investigative strategy has to be determined for each case, as, depending on the structure of the case, the evidence of male DNA on a female victim, or on her clothes, for instance, can also have evidentiary value without microscopic proof for sperm.

Descriptive analyses of differentially expressed genes during larval development of Calliphora vicina (Diptera: Calliphoridae)

The analysis of insect evidence is often used in death investigations as the development of necrophagous insect species can be used to determine a minimum post-mortem interval (PMImin). Usually, the PMImin estimations are based on the age estimation of larvae developed on the corpse. So far, age estimation mostly relies on length or weight measurement of the larvae. These measurements are then compared to species-specific reference growth data obtained from laboratory studies. However, length and weight do not always represent the best developmental markers to provide accurate and precise age estimates for PMImin calculation, especially for post-feeding third-instar larvae which decrease in size. Therefore, more growth markers are required to improve age estimation not only for post-feeding larvae but also for all larval stages. As the analysis of temporally regulated genes proved suitable for age prediction in blow fly pupae, we examined the gene expression patterns of six genes (15_2, 2014192, EcdR, AR, hsp90 and actin) during larval development of Calliphora vicina at three constant temperatures and analysed the effect of a combination of morphological and molecular age markers on statistical models of development. EcdR, AR and 15_2 showed reliable tendencies to classify the post-feeding stage more precisely, and inclusion of gene expression data in models of development improved the statistical fit of the model. Nevertheless, for depicting the first larval stages and for quantifying the onset of the post-feeding stage more accurately, results of this preliminary study should be supported by searching for more informative genes.

Effects of different storage and measuring methods on larval length values for the blow flies (Diptera: Calliphoridae) Lucilia sericata and Calliphora vicina

In forensic entomology, the methods of sampling, killing, and storing entomological samples can affect larval age estimation, and, hence, the estimation of the minimum post-mortem interval. In the existing manuals, there is a certain amount of heterogeneity regarding methods and the recommendations for best practice in forensic entomology are insufficiently validated. This study evaluated three different length-measurement methods for larval stages and examined the influence of different killing and storing methods on the larval length of two forensically important blow flies, Lucilia sericata and Calliphora vicina. The three different measuring methods were a) a ruler with a 0.1mm scale, b) a geometrical micrometer, and c) a computer-aided stereomicroscope. They were used to measure the length of L1-L3C. vicina larvae and detect no significant differences. This supports the view that a simple tool like a geometrical micrometer can produce reliable results in forensic entomology. Newly hatched larvae of L. sericata and C. vicina were killed with hot water (HW) and divided into two equal subsamples. Lengths of all larvae were measured immediately after killing, then every 24h until day 4, and once more after 7days of storage in ≥70%-ethanol. L. sericata larvae only showed significant changes in length in the HW group stored at room temperature. After 4 and 7days of storage, these 24-h- and 72-h-old larvae showed a significant decrease in length compared with those in a fridge at 6°C. This decrease can, however, be considered a negligible natural variation without forensically relevant consequences for larval age estimation of L. sericata samples. For C. vicina, an increase in length was observed over time. This was significant only for younger larvae (24-48h old) stored in 70%-ethanol. This variance in length can lead to a wrong estimation of age; however, only for larvae stored in 70%-ethanol, not for those stored in 96%-ethanol. NOVELTY STATEMENT We examined the influence of different killing and storing methods on two forensically important blow flies, Lucilia sericata and Calliphora vicina. For the latter species we additionally were evaluating three different length measurement methods. The results of both experiments suggest that it is possible to kill and store fly larvae directly in (not hot) ≥70%-ethanol. This simplifies the sampling and storing of fly evidence at the crime scene. We also compared the influence of three different measuring methods for estimating the length of L1-L3 C. vicina larvae by using a) a ruler with a 0,1mm scaling, b) a geometrical micrometer and c) a computer-aided stereomicroscope. No significant differences were detected, supporting the view, that a simple tool like a geometrical micrometer can produce reliable results. This study helps to simplify the sampling and evaluation of entomological evidence and to backup or questioning existing guidelines and best practice recommendations.

Quantitative pteridine fluorescence analysis: A possible age-grading technique for the adult stages of the blow fly Calliphora vicina (Diptera: Calliphoridae)

Age estimation of adult flies could extend the possible window of time for calculating the minimal postmortem interval (PMImin) by means of entomological methods. Currently, this is done by estimating the time required by necrophagous Diptera to reach certain juvenile developmental landmarks, and the method only works until the end of metamorphosis and emergence of the adult fly. Particularly at indoor crime scenes, being able to estimate the age of trapped adult flies would be an important tool with which to extend the calculable PMI beyond the developmental period. Recently, several promising age-dependent morphological and physiological characteristics of adult insects have been investigated in medical and forensic entomology, but the results are still preliminary and restricted to a few species. We examined adults of the forensically relevant blow fly species Calliphora vicina and investigated the fluorescence levels of pteridine, a group of metabolites that accumulates in the eyes during aging. From Day 1 to Day 25 post-emergence, flies were kept at three different temperature regimes (20°C, 25°C, and fluctuating temperatures in the context of a field study) and 12:12 L:D. From Day 1 until Day 7, the fluorescence of pteridine was determined on a daily basis, and thereafter, every three days. The achieved fly age was multiplied with the relevant temperature and converted into accumulated degree-days (ADD). The fluorescence level of pteridine increased linear with increasing ADD (females: R2=0.777; males: R2=0.802). The difference between sexes was significant (p<0.001). Neither head weight nor temperature had an effect on pteridine fluorescence. Because the variation in pteridine fluorescence increased with increasing ADD, it seems favorable to combine several aging methods for more precise results. In context, we emphasize that different body parts of the same specimen can be used to analyze cuticular hydrocarbons (legs), pteridine fluorescence (head/eyes), and gonotrophic stage (female abdomen).

Infanticide and Neonaticide

Infanticide and child homicide are seen in many forms and under widely varying circumstances:

The novel H1N1/swine-origin influenza virus and its implications for autopsy practice

As fatalities attributable to novel H1N1/S-OIV infections are currently rising, an increasing number of autopsies will have to be performed on these cases. In view of this challenge, adequate safety precautions and recommendations for autopsy procedures and the sampling and handling of specimens have to be established. Based on current literature, procedures for daily autopsy practice are proposed, particularly in regard to personal protection equipment, swab usage, and autopsy hygiene.