Environmental factors influencing flight activity of forensically important female blow flies in Central Europe

In forensic entomology, evaluation of a possible delay between a person’s death and insect colonization is crucial. We monitored the seasonal flight activities of the most abundant blow flies in an urban habitat in Frankfurt/Germany based on 152 sampling days between April and October 2017. Thirty-six thousand female specimens of 12 necrophagous taxa were sampled as a possible groundwork for establishing a prediction tool for the activity of certain forensically relevant taxa. The most abundant taxon was Lucilia sericata (n = 19,544), followed by Lucilia caesar (n = 8025), Calliphora vicina (n = 5224), and Lucilia ampullacea (n = 1834). Up to six environmental parameters were statistically significant predictors of fly presence, leading to unique patterns of seasonal and daily activity for all four species. In detail, our analysis proved that L. sericata is a sun-loving, high-summer species that dominates the warmer months and is mostly influenced by mean day temperature. In contrast, L. caesar seems to be a shade-loving species that dominates in autumn resp. late-season and is mainly influenced by mean day temperature and wind speed. The activity of L. ampullacea was highly related to mean day temperature and relative humidity. In contrast to all other species, C. vicina behaved differently, particularly due to its occurrence throughout the entire sampling interval and the higher tolerance limits for the measured abiotic parameters, especially temperature. The present study is groundwork for establishing a prediction tool for the flight and oviposition activity of forensically relevant taxa.

     

Blowfly puparia in a hermetic container: survival under decreasing oxygen conditions

Despite widely accepted standards for sampling and preservation of insect evidence, unrepresentative samples or improperly preserved evidence are encountered frequently in forensic investigations. Here, we report the results of laboratory studies on the survival of Lucilia sericata and Calliphora vomitoria (Diptera: Calliphoridae) intra-puparial forms in hermetic containers, which were stimulated by a recent case. It is demonstrated that the survival of blowfly intra-puparial forms inside airtight containers is dependent on container volume, number of puparia inside, and their age. The survival in both species was found to increase with an increase in the volume of air per 1 mg of puparium per day of development in a hermetic container. Below 0.05 ml of air, no insect survived, and above 0.2 ml of air per 1 mg of puparium per day, survival reached its maximum. These results suggest that blowflies reveal a single, general pattern of survival under decreasing oxygen conditions and that this pattern is a product of number of developing insects, their age and the initial amount of available air. Implications for forensic entomology are discussed.     

Entwicklung von Calliphora vicina unter Nahrungszugabe von Methadon

To estimate the post-mortem interval (PMI), length and developmental state of larvae of several species of blowflies (e.g. Calliphora vicina) can be used. This study deals with the question whether the development of C. vicina is influenced by methadone. Evaluation of the growth factors of the larvae which had been fed on methadone-spiked meat showed on several days significant differences in the length of the larvae compared to the control group. Furthermore, the majority of larvae left the methadone-spiked meat earlier and entered the post-feeding stage up to 1 day earlier than the larvae of the control group. However, the total developmental time only differed from the control group in two out of the four methadone concentrations investigated. At this stage the results of the present study do not allow general statements concerning the determination of the age of larvae which developed under the influence of methadone.     

Nocturnal oviposition behavior of blowflies (Diptera: Calliphoridae) in the southern hemisphere (South Africa and Australia) and its forensic implications

Published research has offered contradictory evidence of the occurrence of nocturnal oviposition by carrion-breeding blowflies, a behavior that can affect the interpretation of forensic estimates of a minimum post mortem interval (_minPMI) by up to 12 hours, depending on latitude and season. The majority of published studies are from the northern hemisphere. Field experiments were conducted in South Africa and Australia that extend observations to species of the southern hemisphere. Various vertebrate carrion was exposed at night in summer under different lunar phases and/or artificial lighting, and in woodland and pasture areas. Three laboratory experiments were also conducted. No nocturnal oviposition occurred outdoors in Berry, Australia, but Lucilia cuprina, Lucilia sericata and Chrysomya megacephala laid eggs outdoors at night in Grahamstown and Durban, South Africa. In laboratory experiments L. sericata, L. cuprina, Chrysomya chloropyga and Chrysomya putoria laid eggs and Calliphora augur deposited larvae under nocturnal conditions. Chrysomya albiceps and C. chloropyga laid eggs in darkness with increasing likelihood as ambient temperature increased. This study shows that nocturnal ovi/larviposition by carrion-breeding blowflies is possible in both South Africa and Australia. The forensic issue is therefore not whether nocturnal oviposition occurs, but rather whether the conditions of a particular case are more or less conducive to it. Circadian rhythms and physiological thresholds (particularly temperature and humidity) appear to act individually and in conjunction to stimulate or inhibit nocturnal laying. The significance of carcass size, freezing and handling of carcasses and comprehensive quantification for experimental design is discussed, and recommendations are made for future laboratory and case scene experiments.     

Predatory behavior of Synthesiomyia nudiseta larvae (Diptera: Muscidae) on several necrophagous blowfly species (Diptera: Calliphoridae)

Intraguild interactions play a prominent role in sarcosaprophagous communities, so intraspecific and interspecific competition phenomena between fly larvae take place. Synthesiomyia nudiseta is a species that has recently been recorded on human corpses in Europe, and it has been cited in forensic cases in Spain and Italy. The mature larvae are known to be facultative predators on necrophagous fly larvae, but their role as predators is unknown. The aim of this paper is to investigate the predatory behavior of S. nudiseta on the most abundant blowfly larvae in sarcosaprophagous communities in southwestern Europe (i.e., Chrysomya albiceps, Lucilia sericata, and Calliphora vicina). Intraspecific and interspecific competition experiments were carried out to study the effect of this species in the necrophagous diptera community. First-instar larvae were placed in plastic cups with the same amount of pig liver (15 g) at different densities (50, 100, 150, and 300 individuals/cup). Mortality in blowflies increased when S. nudiseta larvae were present: up to 98.40% in Ch. albiceps, 89.60% in L. sericata, and 84.93% in C. vicina. Pre-imaginal developmental time and adult wing size were analyzed and compared under each of the intraspecific and interspecific conditions; both variables were affected by competition in all cases. The implications of the presence of S. nudiseta for sarcosaprophagous community dynamics and its effect on the estimation of postmortem interval (PMI) are discussed.

     

Bestimmung des Alters forensisch relevanter Fliegenpuppen

Background

Approximately 50% of the immature development of blowflies takes place in the pupal stage which makes age estimation difficult. This article presents morphological and histological milestones for the pupal development of Calliphora vicina and Lucilia sericata .

Methods

Rearing was carried out at 25°C. Pupae were sampled every 24?h and either weighed or killed. Puparia were removed and pictures were taken. Additionally for analyzing nuclei volumes, adipocytes and histology, pupae were fixed in formalin, sliced and stained with hematoxylin and eosin (7???m, H&E).

Results

Character sheets for external pupal morphology were developed, defining and describing 10 morphological milestones for pupal development. Significant loss of weight was observed during metamorphosis for both species. Larval nuclei showed a reduction of up to 25.3?% of the volume during the first days of histolysis and the number of adipocytes was reduced by up to 52.4?%.

Conclusions

The character sheets show that a combination of morphological characters can be used to estimate age (±?12?h) for the pupae of the blowflies C.?vicina and L.?sericata. Histological changes can confirm or even improve these results.     

Spatio-temporal distribution and habitat preference of necrophagous Calliphoridae based on 160 real cases from Switzerland

Necrophagous blowflies (Diptera: Calliphoridae) are of great importance particularly during investigations of suspicious deaths. Many studies have analyzed the distribution of blowflies based on pig experiments and baited trapping; however, data from real case scenarios are rarely used. In this article, the distribution of blowflies found during investigations of 160 real cases during 1993–2007 in Switzerland is evaluated based on habitat, altitude, and season. Ten species of blowflies were present in 145 out of the 160 cases. The most common species was Calliphora vicina, which occurs throughout the year and was present in 69 % of all cases. Lucilia sericata, Calliphora vomitoria, and L. caesar were identified among the rest of the flies as species of great forensic importance mainly due to their distributional patterns. After a comparison with a similar dataset from Frankfurt, Germany, some surprising differences were determined and discussed. The biggest discrepancies between our dataset and the German dataset were in the occurrences of L. sericata (30 % vs. 86 %, respectively), Phormia regina (5 % vs. 43 %), and L. ampullacea (1 % vs. 45 %). The life-history strategies and intraspecific behavioral variability of blowflies remain understudied, although they can be essential for an unbiased approach during a death investigation. Further research and comparison of occurrence patterns across the area of distribution of blowflies are therefore needed and recommended.

     

Codeine accumulation and elimination in larvae,pupae, and imago of the blowfly <Emphasis Type="Italic">Lucilia sericata</Emphasis> and effects on its development

The aim of this study was to evaluate the reliability of insect larvae as samples for toxicological investigations. For this purpose, larvae of Lucilia sericata were reared on samples of minced pig liver treated with different concentrations of codeine: therapeutic, toxic, and potentially lethal doses. Codeine was detected in all tested larvae, confirming the reliability of these specimens for qualitative toxicology analysis. Furthermore, concentrations measured in larvae were correlated with levels in liver tissue. These observations bring new elements regarding the potential use of opiates concentrations in larvae for estimation of drug levels in human tissues. Morphine and norcodeine, two codeine metabolites, have been also detected at different concentrations depending on the concentration of codeine in pig liver and depending on the substance itself. The effects of codeine on the development of L. sericata were also investigated. Results showed that a 29-h interval bias on the evaluation of the larval stage duration calculated from the larvae weight has to be considered if codeine was present in the larvae substrate. Similarly, a 21-h interval bias on the total duration of development, from egg to imago, has to be considered if codeine was present in the larvae substrate.     

Age estimation during the blow fly intra-puparial period: a qualitative and quantitative approach using micro-computed tomography

Minimum post-mortem interval (_minPMI) estimates often rely on the use of developmental data from blow flies (Diptera: Calliphoridae), which are generally the first colonisers of cadavers and, therefore, exemplar forensic indicators. Developmental data of the intra-puparial period are of particular importance, as it can account for more than half of the developmental duration of the blow fly life cycle. During this period, the insect undergoes metamorphosis inside the opaque, barrel-shaped puparium, formed by the hardening and darkening of the third instar larval cuticle, which shows virtually no external changes until adult emergence. Regrettably, estimates based on the intra-puparial period are severely limited due to the lack of reliable, non-destructive ageing methods and are frequently based solely on qualitative developmental markers. In this study, we use non-destructive micro-computed tomography (micro-CT) for (i) performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of two forensically relevant blow fly species, Calliphora vicina and Lucilia sericata, and (ii) developing a novel and reliable method for estimating insect age in forensic practice. We show that micro-CT provides age-diagnostic qualitative characters for most 10% time intervals of the total intra-puparial period, which can be used over a range of temperatures and with a resolution comparable to more invasive and time-consuming traditional imaging techniques. Moreover, micro-CT can be used to yield a quantitative measure of the development of selected organ systems to be used in combination with qualitative markers. Our results confirm micro-CT as an emerging, powerful tool in medico-legal investigations.

     

Zum Suchtmittelnachweis in Haaren. VII. Untersuchungen bei Methadonpatienten unter konstanter Wirkstoffeinnahme

Hair samples and perspiration collected from long term drug addicts (n = 18) under constant methadone treatment were investigated. Hair segments representing the individual hair growth of 4 weeks were analyzed for methadone, opiates and cocaine by GC/MS. The results obtained from the transdermal collection devices confirmed the presence of drug substances in variable amounts on the skin surface, methadone being the main analyte. The perspiration test period lasted for 4 days. For six persons illicit drugs similar to those in the corresponding hair samples were found. Overall, a poor relationship (r = 0,602) was found between the methadone dose under steady state conditions and the methadone level in hair. The results are discussed in the light of pharmacokinetic aspects, drug uptake by perspiration and forensic interpretation.     

Temperature-dependent appearance of forensically useful flies on carcasses

Flies are frequently used for postmortem interval (PMI) estimations. These estimates are usually based on the age of larval or pupal specimens. However, the age defines only the minimum PMI. In order to move forensic entomology further, a method useful for the estimation of an interval preceding insect appearance on a corpse called the pre-appearance interval (PAI) is needed. Recently, it was demonstrated that the PAI of several carrion beetles is closely related to the temperature prevailing throughout this interval. Hence, it was postulated to estimate PAI from temperature. In order to check premises for using this approach with flies, a test of the relationship between adult or oviposition PAI and temperature was made for nine species of European flies. Data on PAI originated from pig carcasses decomposing under various temperatures. Adult PAI of Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis, Phormia regina, and Stearibia nigriceps and oviposition PAI of S. nigriceps were exponentially related to temperature. Only S. nigriceps revealed a close relationship, demonstrating solid premises for PAI estimation from temperature alone. Adult and oviposition PAI of Calliphora vomitoria and adult PAI of Hydrotaea pilipes were not related to temperature. Adult and oviposition PAI of Lucilia sericata and Lucilia caesar responded similarly, with an abrupt and large increase in a narrow range of low temperatures and no response in a broad range of high temperatures. Probably, different mechanisms form the basis for the response of PAI to temperature in flies colonizing carcasses shortly after death and flies colonizing carcasses later in the decomposition process.     

Comparative fly species composition on indoor and outdoor forensic cases in Malaysia

Forensic entomology refers to the science of collection and analysis of insect evidence in order to determine the minimum time period since death. This study aimed to investigate the occurrence of forensically important flies on 34 human remains referred to Universiti Kebangsaan Malaysia Medical Centre over a period of three years. Entomological specimens were collected at the death scenes and/or during autopsies. Live specimens were reared into adults while preserved specimens were processed for species identification. Five families, seven genera and nine species of flies were identified from human remains. The results of the study showed Chrysomya megacephala (Calliphoridae) maggots occurred on corpses with the highest frequency (70.6%), followed by Ch. rufifacies (Calliphoridae) (44.1%), sarcophagid fly (Sarcophagidae) (38.2%), Synthesiomya nudiseta (Muscidae) (20.6%), Megaselia scalaris (Phoridae) (14.7%), Lucilia cuprina (Calliphoridae) (5.9%), Ch. nigripes (Calliphoridae) (5.9%), Eristalis spp. (Syrphidae) (5.9%) and Hydrotaea spinigera (Muscidae) (2.9%). The greatest fly diversity occurred on remains recovered indoors (eight species) compared to outdoors (three species). Whilst, single and double infestations were common for both indoor and outdoor cases, multiple infestation of up to six species was observed in one of the indoor cases. Although large numbers of fly species were found on human remains, the predominant species were still those of Chrysomya, while S. nudiseta was found only on human remains recovered from indoors. The present study provides additional knowledge in the context of Malaysian forensic entomology and the distribution of forensically important flies which is of relevance to forensic science.     

Applicability of the SNPforID 52-plex panel for human identification and ancestry evaluation in a Brazilian population sample by next-generation sequencing

SNP analysis is of paramount importance in forensic genetics. The development of new technologies in next-generation sequencing allowed processing a large number of markers in various samples simultaneously. Although SNPs are less informative than STRs, they present lower mutation rates and perform better when using degraded samples. Some SNP systems were developed for forensic usage, such as the SNPforID 52-plex, from the SNPforID Consortium, containing 52 bi-allelic SNPs for human identification. In this paper we evaluated the informativeness of this system in a Brazilian population sample (n = 340). DNA libraries were prepared using a customized HaloPlex Target Enrichment System kit (Agilent Technologies, Inc.) and sequenced in the MiSeq Personal Sequencer platform (Illumina Inc.). The methodology presented here allowed the analysis of 51 out of 52 SNPforID markers. Allele frequencies and forensic parameters were estimated, revealing high informativeness: the combined match probability and power of exclusion were 6.48 × 10⁻²¹ and 0.9997, respectively. Population admixture analysis indicates high European contribution (more than 70%) and low Amerindian contribution (less than 10%) in our population, while individual admixture analyses were consistent with the majority of individuals presenting high European contribution. This study demonstrates that the 52-plex kit is suitable for forensic cases in a Brazilian population, presenting results comparable with those obtained using a 16 STR panel.     

Simultaneous analysis of some club drugs in whole blood using solid phase extraction and gas chromatography-mass spectrometry

The use of psychoactive substances to improve social relations and increase body energy, in Rave Culture, has raised many legal and health public concerns, both for illicit trade and consumption. Therefore, forensic toxicology plays an important role in this area, mainly linked to the detection and quantitation of these substances, both in vivo and in post-mortem samples. In fact, at the moment, forensic sciences have been under public authorities’ scrutiny and critical look, due to the increasing attention of the media and public opinion, always applying for the use of scientific knowledge to help solving forensic cases. However, forensic toxicology results are only reliable to solve legal cases if all the analytical methodologies used are appropriately validated.In this work, a methodology for the extraction and analysis of 7-aminoflunitrazepam, buprenorphine, flunitrazepam, ketamine, methadone, phencyclidine (PCP) and d-propoxyphene was developed for whole blood samples, with solid phase extraction (SPE), using OASIS^® MCX SPE columns, and gas chromatography coupled to mass spectrometry.The procedure presented here proved to be reliable, specific, selective and sensitive, with good LODs and LOQs and good precision.The adoption of a SPE procedure with an automatic SPE extraction device, allowed an increased level of automation in sample treatment, being contemporarily less time-consuming, increasing productiveness, and allowing good recovery and appropriate selectivity being, also, simple and reproducible. The simultaneous detection and quantitation of all compounds by the same extraction and detection methodology is crucial and has a great potential for forensic toxicology and clinical analysis.     

Same,same but different!—matching entomological traces to a human food source by stable isotope analysis

Blow flies are important tools in forensic entomology, as they feed and develop on human bodies. This fact can be used to estimate the minimum post mortem interval (PMI_min), by classifying the age of the immature stages found on corpses. The capacity to also be able to classify the age of adult flies, or even empty puparia, could increase the timeframe for a PMI_min. However, the association of older developmental stages, such as adult flies or their empty cases, with a human cadaver may be challenged, as such specimens could stem from another food source. Analyzing the stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes in such specimens could be helpful here, as the isotope signatures reflect those of the food sources. We compared the δ¹⁵N and δ¹³C signatures of tissue from humans and 12 additional species by elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). We did the same for adult flies and empty puparia of the blow fly Lucilia sericata, which were derived from juvenile stages developed on tissues from the mentioned species. The isotope signatures for the adult flies and their empty puparia linearly mirrored the isotope signatures for the respective tissues on which they developed as larvae. Results such as these are useful to indicate the flies’ nutritional history, but they do not provide the same kind of evidence that a DNA analysis would. As individual dietary habits, global distribution and environmental effects can influence isotope ratios, we strongly recommend to analyzing entomological traces and muscle tissues of potential donors or for other food sources.

     

Molecular identification of carrion-breeding scuttle flies (Diptera: Phoridae) using COI barcodes

Entomological evidence is often used in forensic cases for post-mortem interval (PMI) calculation. The most dominant species present on a corpse are typically blowflies. However, several cases have been reported where access to a corpse has been restricted for blowflies (e.g., on a buried or wrapped cadavers) but species of the family Phoridae were abundant. It has also been reported that some phorid species that exploit human corpses may also feature in cases of myiasis acquired ante-mortem. In all these cases, they may provide decisive evidence. As for blowflies, the precise identification of a phorid species collected from a corpse is necessary when estimating the PMI. Since morphological determination is often hampered due to similar characteristics especially in the larval and pupal stage, we used DNA-based methods to identify six phorid species (Megaselia scalaris, Megaselia giraudii, Megaselia abdita, Megaselia rufipes, Conicera tibialis, and Puliciphora borinquenensis) on the molecular level. We focused on a 658-bp-long region of the cytochrome oxidase I gene (COI), the most common molecular marker in forensic entomology. The amplified fragment is also used in DNA barcode approaches and was found to be suitable for identification of a wide range of insect taxa. The present study demonstrates that this region is also sufficient to distinguish between several species of scuttle flies.     

Estimation of stature from lengths of index and ring fingers in a North-eastern Indian population

Forensic anthropology involves identification of human remains in a legal situation. Along with the other three parameters of forensic anthropology, i.e., age, sex and ethnicity, stature estimation is an essential element of medico-legal investigations when identifications of unknown dismembered remains are involved. The present study was conducted with an aim to find the correlation between stature and the lengths of the index and ring fingers and to derive linear and multiple regression models for estimation of adult stature from the index finger and ring finger measurements. The subjects chosen for the study were composed of adult individuals belonging to the “Rajbanshi”, an ethnic community of North-eastern India. A total of 500 adults (250 males and 250 females) aged between 18 years and 60 years took part in the study. All the measurements were taken using standard techniques. Stature, index finger length and ring finger length were significantly longer in the males than the females. The correlation coefficient (r) between stature and finger lengths was observed to be positive and statistically significant. Linear and multiple regression models were derived for estimation of stature from a length of index and ring fingers. The predictive accuracy of stature estimation was higher for the females than the males. It was observed that the values of the correlation coefficient (r) and the coefficient of determination (R²) increased in multiple regression models when compared to the linear regression models. The study also focuses on the usability of sex-specific regression models in forensic casework. The study concludes that such regression models, as those derived in the present work, can be of significant utility in the estimation of stature.     

Some tips on age estimation using DNA methylation in saliva samples as an index across the Japanese and Indonesian ethnicities

Age estimation of unidentified bodies is of marked importance in forensic medicine. In previous studies, the analysis of DNA methylation in body fluids led to the identification of several age-related CpG sites in genes such as EDARADD and FHL2. However, limited information is available on whether interethnic differences may affect the age prediction results. In the present study, we examined the effect of ethnicity on the age prediction method based on methylation scores, which were determined via methylation-sensitive high-resolution melting. We found that there was a significant difference in methylation scores between Japanese and Indonesian participants of early 20 s group, and that the nationality coefficient was significant for age estimation when applying the existing method for the analysis of the methylation status of EDARADD and FHL2. This suggests that when using certain biochemical indicators as a predictor of age, the effects of ethnicity on DNA methylation should be considered to improve the accuracy of the estimation.     

Intravenous methadone application as a serious risk factor for an overdose death: methadone-related fatalities in Hamburg from 2007 to 2012

Methadone plays an increasing role in drug-related deaths in Hamburg. To find out whether intravenous application of methadone plays a relevant role in methadone-related deaths, body fluids of all methadone-positive cases (n?=?130) and three buprenorphine-positive cases where a urine sample was available (n?=?58?+?3) were investigated for disaccharides (sucrose and lactose as markers for intravenous methadone abuse). Sixty-four percent of the urine samples of the methadone cases showed positive results for disaccharides (22 times sucrose alone, range 2 to >1,000 mg/L; 6 times lactose and sucrose; and 9 times lactose alone, range 22 to 382 mg/L). The three buprenorphine cases showed positive results for lactose in urine. In blood, it was not possible to detect any disaccharides. Of the 116 fatal methadone intoxications, 49 % were under opiate maintenance treatment (OMT) at the point of death (A-OMT), 30 % were never in OMT (N-OMT) and 21 % were formerly in an OMT, but not at the point of death (F-OMT). Of the deceased in the OMT group, 12 % (n?=?7) died within the first 2 weeks of treatment, six of them within the first week. Overall, intravenous abuse of methadone plays a relevant role in methadone-related fatal cases of substituted patients and of drug consumers not in therapy. Thus, it is necessary that therapists keep to the statutory regulations and give take-home doses only after at least 6 months of successful therapy and when there is no suspicion of intravenous abuse.     

Molecular pathology of brain edema after severe burns in forensic autopsy cases with special regard to the importance of reference gene selection

Brain edema is believed to be linked to high mortality incidence after severe burns. The present study investigated the molecular pathology of brain damage and responses involving brain edema in forensic autopsy cases of fire fatality (n?=?55) compared with sudden cardiac death (n?=?11), mechanical asphyxia (n?=?13), and non-brain injury cases (n?=?22). Postmortem mRNA and immunohistochemical expressions of aquaporins (AQPs), claudin5 (CLDN5), and matrix metalloproteinases (MMPs) were examined. Prolonged deaths due to severe burns showed an increase in brain water content, but relative mRNA quantification, using different normalization methods, showed inconsistent results: in prolonged deaths due to severe burns, higher expression levels were detected for all markers when three previously validated reference genes, PES1, POLR2A, and IPO8, were used for normalization, higher for AQP1 and MMP9 when GAPDH alone was used for normalization and higher for MMP9, but lower for MMP2 when B2M alone was used for normalization. Additionally, when B2M alone was used for normalization, higher expression of AQP4 was detected in acute fire deaths. Furthermore, the expression stability values of these five reference genes calculated by geNorm demonstrated that B2M was the least stable one, followed by GAPDH. In immunostaining, only AQP1 and MMP9 showed differences among the causes of death: they were evident in most prolonged deaths due to severe burns. These findings suggest that systematic analysis of gene expressions using real-time PCR might be a useful procedure in forensic death investigation, and validation of reference genes is crucial.