Successive Protein Extraction Using Hydroxylamine to Increase the Depth of Proteome Coverage in Fresh, Forensic, and Archaeological Bones.

Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, particularly collagen-containing tissues such as bones and teeth. Here, we present the application of a chemical digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical, and archaeological samples. We describe and discuss two protocols: one that uses hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another that applies hydroxylamine directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including an increase in the sequence coverage and number of peptides detected in modern and archaeological samples and an increase in the number of proteins identified in archaeological samples. By targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights, considering both fresh and ancient mineralized samples.

A sudden death, an aortic rupture, and an unexpected cause: a report about suspected child abuse.

All unexpected deaths of children require an autopsy to determine the cause of death. In cases of aortic rupture, the immediate cause of death is easily identified at autopsy. Although the majority of aortic ruptures are caused by high-energy trauma, other causes should not be missed.We present and discuss the case of a 29-month-old child who died suddenly at home. Her recent medical history and the ecchymotic lesions observed on external examination of the body appeared potentially suspicious of physical abuse. The autopsy concluded that death was due to complete rupture of the abdominal aorta with associated vertebral disjunction. At first glance, the overall forensic picture could suggest a traumatic death. However, careful inspection of the retroperitoneum revealed a discrete atypical mass of infiltrative tissue within the hematoma. Histopathological examinations confirmed tumor proliferation of the soft tissues, triggering vascular and spinal injuries. Other paraneoplastic elements or metastases were ultimately revealed (orbital and subcutaneous). Overall, this was a rare and fatal case of abdominal aortic rupture induced by tumors. Due to the mechanisms and the forces needed to cause vertebral dislocations and aortic rupture, the combination of the two is highly suggestive of child abuse when an accidental traumatic history is absent or inconsistent with the injuries. Nevertheless, this case illustrates the importance of a systematic and rigorous forensic examination, rather than ignoring other possible diagnoses.

Study of Root Transparency in Different Postmortem Intervals Using Scanning Electron Microscopy.

In the fields of forensics, the identification of human remains is a recurrent problem. The estimated age at death is one of the copious criteria to be evaluated. In adult teeth, the height of the root dentin transparency is used to estimate age. However, in archaeological material, this phenomenon appears inconstant. The aim of this work was to observe the structural modifications of the sclerotic dentin in the teeth for different postmortem intervals. The study included two parts (retrospective and prospective study) with 21 human monoradicular teeth, from bodies donated to medical science with postmortem intervals (PMIs) of 0, 1, 2 and 5 years and archeological excavation. After inclusion based on resin, section and polishing, the samples were analyzed with a scanning electron microscope (SEM) JSM-7800F®, and the procedure was completed via a semiquantitative analysis of calcium and phosphorus using EDX microanalysis. The analysis showed the existence of tubular and chemical modifications of sclerotic dentin at different PMIs. Our SEM study allowed us to observe a difference in tubule aspects linked to an increased PMI: the loss of peritubular collar and the lumen obstruction of tubules with a hyperdense material. Microanalysis highlighted variations in phosphocalcic ratios among the different groups, especially in the pulp area and the canine. Our hypotheses that explain these differences are based on the postmortem modifications of the crystals of the mineral phase of sclerotic dentin under the influence of chemical and/or bacterial action.

Interpretation of hair and nails findings in an infant death case related to maternal addiction to tramadol.

An 11-month-old boy was found dead. Autopsy findings (cyanosis and polyvisceral congestion) and blood tramadol (TR) concentration of 6240 µg/L were consistent with an acute TR intoxication. In this poisoning situation, owing to the mother's statements (TR addiction leading to daily TR-orange juice mixture preparation accidentally used for the baby bottle preparation by the mother's partner), and the question of possible previous TR administrations to the infant, hair and/or nails (infant, mother, partner, 6-year-old sister) analysis was performed. Hair (2-cm-long hair segments from proximal [S1] to distal [S3]) and nails concentrations (pg/mg; nd: not detected) were as follows: Infant (hair: TR 1420 [S1], 1622 [S2], 2736 [S3]; O-DMT 16-38; N-DMT 34-100 [TR in significant quantities in the hair decontamination bath]-toenails: TR 584; O-DMT 8; N-DMT 15), mother (hair: TR 2340 [S1], 2150 [S2], 2500 [S3]; O-DMT 704-1170; N-DMT 827-1360), mother's partner (fingernails: TR 72; O-DMT nd; N-DMT nd) and sister (hair: TR 261 [S1], 524 [S2]; O-DMT 15 [S1], 16 [S2]; N-DMT 20 [S1], 38 [S2]). Metabolite ratio (infant and sister hair) was comparable to those observed in hair of pharmaceutical industry employees manufacturing tramadol. TR in washing baths, low observed nail concentrations (infant and partner) confirm (i) TR-related mother's addiction and (ii) external contamination issues (TR in sweat of the child at the time of death and in living environment) to explain the infant's keratinized samples results. This case report illustrates the interest of analyzing keratinized matrices of the whole family in such a situation.

In the case of extensively putrefied bodies, the analysis of entomological samples may support and complement the toxicological results obtained with other alternative matrices.

Entomotoxicological analysis is not part of routine toxicological analysis. This work aims to present two cases to illustrate the potential of entomological samples as complementary matrices to identify substances in cases of advanced putrefaction. (Case#1) A woman wasexhumed after 14 months to ascertain the exact cause of death. She died after six weeks of hospitalization because of intestinal ischemia followed by multiorgan failure. (Case#2) The corpse of a woman, known to have a psychiatric disorder, was discovered in her apartment. The state of decomposition of the body was consistent with a post-mortem period of several weeks (approximately 6 weeks). Toxicological investigations were performed in the biological and entomological samples of case#1 (hair, adipocere, brain, and pupae) and of case#2 (hair, bone, flies, and pupae) using liquid chromatography with high-resolution mass spectrometry and tandem mass spectrometry detection methods. In case#1, several drugs and metabolites were detected. In particular, the pupae analyses allowed the objectification of morphine administration, whereas morphine was only found in adipocere, but not in hair nor in brain. In case#2, the pupae analyses allowed the detection of three metabolites of quetiapine, and the flies analyses allowed the detection of valpromide, which was only detected in hair. In conclusion, the pupae and flies analyses in these two cases complemented the results obtained in the other alternative biological samples, which may guide hypotheses about the possible causes of death. Nevertheless, additional data and case reports would be of benefit to assess the value of entomotoxicology in routine forensic investigations.

Automated age-at-death estimation by cementochronology: Essential application or additional complication?

It has been repeatedly acknowledged that age-at-death estimation based on dental cementum represents a partial and time-consuming method that hinders adoption of this histological approach. User-friendly micrograph analysis represents a growing request of cementochronology. This article evaluates the feasibility of using a module to accurately quantify cementum deposits and compares the module's performance to that of a human expert. On a dental collection (n = 200) of known-age individuals, precision and accuracy of estimates performed by a developed program (101 count/tooth; n = 20,200 counts) were compared to counts performed manually (5 counts/tooth; n = 975 counts). Reliability of the software and agreement between the two approaches were assessed by intraclass correlation coefficient and Bland Altman analysis. The automated module produced reliable and reproducible counts with a higher global precision than the human expert. Although the software is slightly more precise, it shows higher sensitivity to taphonomic damages and does not avoid the trajectory effect described for age-at-death estimation in adults. Likewise, for human counts, global accuracy is acceptable, but underestimations increase with age. The quantification of the agreement between the two approaches shows a minor bias, and 94% of individuals fall within the intervals of agreement. Automation gives an impression of objectivity even though the region of interest, profile position and parameters are defined manually. The automated system may represent a time-saving module that can allow an increase in sample size, which is particularly stimulating for population-based studies.

Traumatic rectovaginal fistula after sexual intercourse following a non-consensual anal penetration: a case report and a review of the literature.

Rectovaginal fistulas are rare genital lesions, often due to obstetric causes. More rarely they are a consequence of sexual intercourse. We present the case of a 36-year-old woman who presented to a gynecological emergency department with pelvic pain and feces coming from the vagina. This symptomatology appeared after non-consensual anal intercourse following consensual penile vaginal penetration. She was treated for a perforation of the rectovaginal septum. On the basis of our results, we reviewed the cases published in the literature, with a summary of the elements that may favor the appearance of this lesion, such as "virginity", obstetrics and gynecological history, or coitus position, its classification by size and localization, and the therapeutic indications (suture repair of the wall or a colostomy). Better knowledge of this type of lesion would improve the practice of forensic pathologists for screening and management.

Effect of density and species preferences on collective choices: an experimental study on maggot aggregation behaviours.

Collective decisions have been extensively studied in arthropods, but they remain poorly understood in heterospecific groups. This study was designed to (1) assess the collective behaviours of blow fly larvae (Diptera: Calliphoridae) in groups varying in density and species composition, and (2) relate them to the costs and benefits of aggregating on fresh or decomposed food. First, experiments testing conspecific groups of Lucilia sericata and Calliphora vicina larvae, two species feeding at the same time on fresh carcasses, demonstrated decreases in growth and survival on rotten beef liver compared with fresh liver. However, mixing species together reduced this adverse impact of decomposition by increasing the mass of emerged adults. Second, larval groups were observed in binary choice tests between fresh and rotten liver (i.e. optimal and sub-optimal food sources). The results showed that larvae interacted with each other and that these interactions influenced their food preferences. We observed that (1) larvae were able to collectively choose the optimal food, (2) their choice accuracy increased with larval density and (3) the presence of another species induced a reversal in larval preference towards rotten food. These results highlight the ubiquity of collective decision properties in gregarious insects. They also reveal an unexpected effect of interspecific association, suggesting the colonization of new resources through a developmental niche construction.

Benefits of heterospecific aggregation on necromass: influence of temperature, group density, and composition on fitness-related traits.

Necrophagous blowflies (Diptera: Calliphoridae) such as Calliphora vicina, a cold-tolerant species, and Lucilia sericata, a warm-adapted species, are pioneer carrion-breeder. Although these two species have different temperature preferences, larvae aggregate actively and often feed simultaneously on carrion. The hypothesis to be tested was that L. sericata benefits from the association with C. vicina at lower temperatures (i.e., 15 °C) and that C. vicina derives greater benefits from this association at higher temperatures (i.e., 28 °C). Therefore, both species were raised at these two constant temperatures from first instars to adults under three different conditions: monospecific low-density, monospecific high-density, and heterospecific high-density. The time until larval migration, surface area of puparia, and survival rates were determined for each condition. Differences between these fitness-related traits were found between species, temperatures, group densities, and species compositions. At 28 °C, C. vicina larvae bred in heterospecific groups migrated significantly earlier and in higher numbers than that under same density conspecific conditions, with a lower mortality rate. At 15 °C, both species benefited from high-density heterospecific associations, expressed by faster development and larger puparia. In conclusion, necrophagous larvae benefited from heterospecific aggregations at suboptimal temperatures by adapting their migration time to that of the faster species. Since temperature changes throughout the day and over the year, the beneficiary of such a collective association also changes. The costs involved and deviations to the temperature-size rule highlight the complexity of the carrion ecosystem.

Medico-legal considerations for Morel-Lavallée lesions.

The Morel-Lavallée lesion is an infrequent traumatic lesion that the forensic physician may need to evaluate during examination of a victim. Using a review of the literature and an illustrative case, the objective of this paper is to discuss the medico-legal issues (accountability, functional recovery and healing times, and aftermath) associated with Morel-Lavallée lesions. We describe the case of a 24-year-old motorcycle driver who was hit by a car. Clinically, the right lumbar region had a large 16 × 15 cm ecchymosis with subcutaneous fluid swelling found by palpation. A body scan revealed a Morel-Lavallée lesion among other lesions. On day 7, its thickness was decreased by half. Morel-Lavallée lesions are specifically induced by shearing force with moderate-to-high kinetics in an anatomical area with an underlying fascia. The associated functional disability is globally moderate, but a large lesion or recurrences can extend the time needed to recover. Apart from some exceptions, care without any delay in diagnosis and treatment allows evolution of the lesion without functional consequences, but these lesions are frequently unnoticed in the initial phase.

Determination of the age of bruises using a bilirubinometer.

The dating of bruises can be of crucial interest in clinical forensic medicine, but the macroscopic or photographic evaluation of a bruise has not been regarded as reliable. Several methods have thus been regularly evaluated for this purpose, with reflectance spectrometry yielding promising results. The objective of this study is to evaluate the value of a bilirubinometer as a tool for dating bruises on living victims. A clinical follow-up was carried out on patients presenting a bruise with a known onset. Using a bilirubinometer, we obtained daily measurements of the bruise (bili-bruise) and of healthy skin (bili-skin). Potential confounding factors were collected: age, sex, body mass index, trauma mechanism and Fitzpatrick skin phototype. We followed 20 patients for a total of 88 measurements of bruises. Bili-skin values showed significant differences according to skin phototype. Differences between the bili-bruise and bili-skin values (Δ-bili) followed an increase phase, peaking between 3 and 5 days, and then there was a decrease phase. No significant Δ-bili value differences were observed based on the suspected confounding factors. Our results are in favour of a peak Δ-bili value generally at day 4 or 5 post-trauma. Notably, decreasing values were not observed before day 3. Decreasing Δ-bili values would then indicate a bruise resulting from an injury formed at least 3 days before the first measurement. Complementary work confirming such data would enable improvement of the performance of bruise dating in forensic medicine.

Age at death estimation by cementochronology: Too precise to be true or too precise to be accurate?

OBJECTIVES: Cementochronology based on annual deposition of acellular cementum is acknowledged for its superior performance for estimating age-at-death but is also disregarded because of its suspicious effectiveness. This article aims to provide a standardized framework for the validation of the technique and to define and test cementochronology's performance and limitations. MATERIALS AND METHODS: To determine the boundaries of the cementum aging technique, we applied a certified protocol on a sample of 200 healthy canines from individuals of known age, sex, postmortem, and inhumation intervals from anthropological and anatomical collections. We scored readability and preservation of cementum and measured the agreement between estimates, i.e., the precision, and assessed the quality of the accordance between estimates and chronological age, i.e., the accuracy. To investigate the applicability on ancient material, 200 additional canines extracted from archeological assemblages were included. Accuracy and precision were analyzed for each age group in considering postmortem intervals and taphonomical conditions. RESULTS: A strong correlation was found between chronological age and estimates (r = .927; p = .000) but results reveal an association between readability of incremental lines and chronological age (p < .05) and a notable difference in both precision and accuracy between individuals under and over 50 years. Results also demonstrate that taphonomy can be a serious obstacle increasing imprecision by a factor of three. DISCUSSION: Improperly adopted, cementochronology can lead to precise but inaccurate estimations. If methodological, physiological, and taphonomical factors are taken into account, then, and only then, cementochronology will serve as a versatile and powerful tool for age-at-death estimation.

The maggot, the ethologist and the forensic entomologist: Sociality and thermoregulation in necrophagous larvae.

Necrophagous insects are mostly known through forensic entomology. Indeed, experimental data investigating the effect of temperature on larval development underlies post-mortem interval estimations. However, such developmental studies rarely considered the behavior of maggots. In contrast, previous results supposed that calliphoridae larvae use behavioral strategies to optimize their development on carcasses. To test this idea, we analyzed the trade-off between thermal regulation (individual thermal preferences) and social behavior (aggregation) in Lucilia sericata larvae. The first set of experiments analyzed the behavior of third instars in response to thermal changes in their environment. The results demonstrated a clear thermoregulation behavior, supporting the assumption that larvae continuously move to reach a suitable internal temperature. The second set of experiments focused on the trade-off between thermal optimization and aggregation. The results showed a constant search for congeners and an attractiveness of aggregates, sometimes to the detriment of thermal optimization. Together, these results demonstrate a balance between behavioral thermoregulation and social strategies, two significant mechanisms for developmental optimization in necrophagous larvae. In conclusion, these findings highlights unexpected (social) strategies to cope with ephemeral resource and high selection pressure. They also raise important questions for forensic entomology.

Mixed-species aggregations in arthropods.

This review offers the first synthesis of the research on mixed-species groupings of arthropods and highlights the behavioral and evolutionary questions raised by such behavior. Mixed-species groups are commonly found in mammals and birds. Such groups are also observed in a large range of arthropod taxa independent of their level of sociality. Several examples are presented to highlight the mechanisms underlying such groupings, particularly the evidence for phylogenetic proximity between members that promotes cross-species recognition. The advantages offered by such aggregates are described and discussed. These advantages can be attributed to the increase in group size and could be identical to those of nonmixed groupings, but competition-cooperation dynamics might also be involved, and such effects may differ between homo- and heterospecific groups. We discuss three extreme cases of interspecific recognition that are likely involved in mixed-species groups as vectors for cross-species aggregation: tolerance behavior between two social species, one-way mechanism in which one species is attractive to others and two-way mechanism of mutual attraction. As shown in this review, the study of mixed-species groups offers biologists an interesting way to explore the frontiers of cooperation-competition, including the process of sympatric speciation.

Temperature: the weak point of forensic entomology.

Measuring temperature is a key factor in forensic entomology. While noting factors to consider for a posteriori temperature estimation, many studies lack detailed methods or general rules allowing their integration into insect development-time calculations. This article proposes tools for determining the adequacy of weather station temperature datasets versus the local temperature experienced by carrion breeders. The idea is to start from a local scale (i.e., the cadaver) and gradually move to larger scales: at each step, the temperature can be increased, decreased or smoothed by environmental or biological factors. While a one-size-fits-all solution is not feasible for a complex and sensitive issue such as forensic meteorology, this checklist increases the reliability of minimum post-mortem interval (PMImin) estimation and the traceability of the proposed assumption.

Histological dating of subdural hematoma in infants.

BACKGROUND: After infant deaths due to non-accidental head injury (NAHI) with subdural hematoma (SDH), the magistrates ask experts to date the traumatic event. To do so, the expert only has tools based on adult series of NAHI. We aimed to develop an SDH dating system applicable to infants aged under 3 years. METHODS AND RESULTS: We studied a retrospective multicenter collection of 235 infants who died between the ages of 0 and 36 months, diagnosed with SDH by forensic pathological examination and with known posttraumatic interval (PTI). Two pathologists assessed blindly and independently 12 histomorphological criteria relating to the clot and 14 relating to the dura mater in 73 victims (31 girls, 42 boys) whose median age was 3.8 months. Histopathological changes were significantly correlated with PTI for the appearance of red blood cells (RBCs) and the presence or absence of siderophages, and regarding the dura mater, the quantity of lymphocytes, macrophages, and siderophages; presence or absence of hematoidin deposits; collagen and fibroblast formation; neomembrane thickness; and presence or absence of neovascularization. Dating systems for SDH in adults are not applicable to infants. Notably, neomembrane of organized connective tissue is formed earlier in infants than in adults. CONCLUSION: Our dating system improves the precision and reliability of forensic pathological expert examination of NAHI, particularly for age estimation of SDH in infants. However, the expert can only define a time interval. Histopathology is indispensable to detect repetitive trauma.

Communication in necrophagous Diptera larvae: interspecific effect of cues left behind by maggots and implications in their aggregation.

Necrophagous Calliphoridae breed in vertebrate carrion. Their larvae aggregate and form large masses of individuals. These aggregated larvae can reach adulthood faster than scattered larvae, increasing their chances of survival. Furthermore, the gathering of larvae of different species suggests possible interspecific aggregation vectors. In this context, the effect of larval ground-left cues on larvae of Calliphora vomitoria and Lucilia sericata was studied. We used video tracking to follow larvae placed in binary choice tests. We observed (1) a preference of both species for a side marked by conspecific or heterospecific larvae compared to an unmarked side, (2) a preference of L. sericata larvae for a conspecific-marked side compared to a heterospecific-marked side but only at high concentration of cues and (3) a preference of both species for the side marked by the greater number of larvae. These results demonstrate that larvae leave a mark locally which is retentive, has an interspecific range, has an effect proportional to its intensity and whose strength varies depending on the emitting species. According to the self-organization theory, this mark could enhance larval gathering and promote interspecific aggregations. While not yet demonstrated, an interspecific Allee effect could explain the interspecific association of necrophagous calliphorid larvae.

To eat or get heat: Behavioral trade-offs between thermoregulation and feeding in gregarious necrophagous larvae.

The thermoregulation behavior of Lucilia sericata larvae (Diptera: Calliphoridae), a necrophagous species that feeds on vertebrate cadavers, was investigated. These larvae require high heat incomes to develop, and can elevate temperatures by forming large aggregates. We hypothesized that L. sericata larvae should continue to feed at temperatures up to 38 °C, which can be reached inside larval masses. Thermal regulation behavior such as movement between a hot food spot and colder areas was also postulated. The hypotheses were tested by tracking for 1 h the activity of single, starved third instar larvae in a Petri dish containing 1 food spot (FS) that was heated to a constant temperature of 25 °C, 34 °C or 38 °C with an ambient temperature of 25 °C. The influence of previous conspecific activity in the food on larval behavior was also tested. The crops of larvae were dissected to monitor food content in the digestive systems. Based on relative crop measurements, larvae fed at all food temperatures, but temperature strongly affected larval behavior and kinematics. The total time spent by larvae in FS and the duration of each stay decreased at high FS temperature. Previous activity of conspecifics in the food slightly increased the time spent by larvae in FS and also decreased the average distance to FS. Therefore, necrophagous L. sericata larvae likely thermoregulate during normal feeding activities by adjusting to local fluctuations in temperature, particularly inside maggot masses. By maintaining a steady internal body temperature, larvae likely reduce their development time.

Fatal Overdose of Gamma-hydroxybutyrate Acid After Ingestion of 1,4-Butanediol.

We report a case of fatal intoxication from 1,4-butanediol (1,4-BD), which was ingested by a young and "naïve" gamma-hydroxybutyrate (GHB) consumer during a party with the co-ingestion of alcohol, cannabis, and methylene-dioxy-methamphetamine. The following drug concentrations were found using gas chromatography coupled with mass spectrometry on autopsy samples and on a cup and a glass found at the scene: 20,350 mg/L (bottle) for 1,4-BD; 1020 mg/L (femoral blood), 3380 mg/L (cardiac blood), 47,280 mg/L (gastric content), and 570 mg/L (vitreous humor) for GHB. The concentration of GHB is difficult to interpret in forensic cases due to the possibility of an endogenous production of GHB. The variable tolerance of the user may also modify the peri- and postmortem GHB concentrations. This case underscores the need to have many different sources of toxicology samples analyzed to avoid the hypothesis of endogenous production of GHB.

Is bone analysis with µCT useful for short postmortem interval estimation?

The evaluation of the postmortem interval (PMI) is an important consideration in forensic medicine. The time between death and the discovery thereof is extremely important, not only in terms of the legal process, but also for the course of the investigation. At the time of the discovery of human bones, the evaluation of the PMI is extremely difficult. To date, there is no really effective tool for this evaluation. The objective of our study is to characterize the architectural modifications of human bones using micro-tomography (µCT) in the postmortem period. Ten bone samples were extracted from a single fresh human cranial vault (no freezing, no embalming) from a body donation. The samples were conserved in a controlled environment (a laboratory hood at 20 °C with 60% humidity) for 10 weeks. Every 2 weeks after the death and the sample extraction from the vault, the samples were analyzed with µCT (resolution 10 µm, Bruker HR1172 ®). The 3D analysis focused on general 3D data related to the trabeculae: the total volume (TV), the bone volume (BV), and the bone surface (BS), as well as specific trabeculae data; namely: the number of trabeculae (TbN), trabecular thickness (TbTh), the average distance between the trabeculae (TbSp), and the trabecular pattern factor (TbPf). Each sample was observed separately and compared to itself over time; no comparisons among samples were made. We used a linear mixed model for repeated measures (an unstructured covariance pattern model) and post hoc comparisons between each consecutive time using linear contrast with an SAS software package, release 9.3 (SAS Institute, Cary, NC). We showed a statistical difference for BS/BV (p = 0.046) over time, with a significant decrease during the first 2 weeks (p = 0.003; mean decrease, - 0.99, 95% CI [- 1.61; - 0.36]), while TbSp (p < 0.0001) showed a significant increase during the first 2 weeks (p < 0.0001; mean increase, 0.022, 95% CI, [- 0.016; - 0.028]).Our study highlighted architectural trabecular modifications on postmortem bones using the µCT. These modifications occurred at an early stage, during the first 2 weeks of conservation. Complementary work will need to be performed to allow for better understanding of the biological phenomena observed and to create databases of osseous taphonomy, starting with various individuals, mediums of conservation, types of bone, and with varying techniques for analysis.