The Optimal Combination of Cartilage Source and Apparatus for Long‐Term In Vitro Chondrocyte Viability Analysis

Abstract: Most studies of long‐term chondrocytes survival were for tissue banks. They showed a gradual reduction in the viable chondrocytes percentage as a function of time and ambient temperature, but the samples were harvested under optimal conditions. The aim of our study was to determine the most reliable combination of cartilage source and assay for the in vitro postmortem chondrocyte viability analysis in the conditions that imitate a dead body. Osteochondral cylinders were procured from femoral condyles and talar trochleas of three male donors and stored in the cell culture media at 4 ± 2°C and 23 ± 2°C. The samples were analyzed by a cell viability analyzer and a confocal laser scanning microscope (CLSM) initially 24–36 h after death and then in 4‐week intervals. The results reconfirmed the significant influence of time (p = 0.0002), but not of the temperature (p = 0.237). The largest reproducibility was presented for the knee joint and the CLSM.     

The Effect of Soft Tissue on Temperature Estimation from Burnt Bone Using Fourier Transform Infrared Spectroscopy

This study investigated the effect of soft tissue and different exposure times on the prediction of burning temperatures of bone when using Fourier transform infrared spectroscopy (FTIR). Ovis aries rib bones were burnt at different temperatures and for varying time intervals. Results of a linear regression analysis indicated that burn temperatures can be predicted with a standard error of ±70°C from defleshed bone spectra. Exposure time does not have a significant impact on prediction accuracy. The presence of soft tissue has a significant impact on heat‐induced changes of the bone matrix in low (<300°C) as well as high temperatures (>800°C), slowing down combustion in the former and accelerating it in the latter (p < 0.05). At medium temperatures, no significant difference was noted. These results provide forensic investigators a new perspective with which to interpret the results of crystallinity measures derived from burnt bone.     

Forensic Use of A Subtropical Blowfly: The First Case Indicating Minimum Postmortem Interval (mPMI) in Southern Brazil and First Record of Sarconesia Chlorogaster from a Human Corpse

Southern Brazil is unique due to its subtropical climate. Here, we report on the first forensic entomology case and the first record of Sarconesia chlorogaster (Wiedemann) in a human corpse in this region. Flies' samples were collected from a body indoors at 20°C. Four species were found, but only Chrysomya albiceps (Wiedemann) and S. chlorogaster were used to estimate the minimum postmortem interval (mPMI). The mPMI was calculated using accumulated degree hour (ADH) and developmental time. The S. chlorogaster puparium collected was light in color, so we used an experiment to establish a more accurate estimate for time since initiation of pupation where we found full tanning after 3 h. Development of C. albiceps at 20°C to the end of the third instar is 7.4 days. The mPMI based on S. chlorogaster (developmental time until the third instar with no more than 3 h of pupae development) was 7.6 days.     

The Role of Vitreous Magnesium Quantification in Estimating the Postmortem Interval

The use of magnesium as a parameter to estimate the time of death is controversial. In 32 traumatic deaths with known postmortem intervals (PMIs), small quantities of vitreous humor (VH) were sampled repetitively every 3 h until 24 h postmortem. The bodies were kept at the constant ambient temperature of 20°C (68°F). The concentrations of magnesium were in the range of 0.47–1.15 mM. A statistically significant correlation of the concentration of magnesium with the PMI was observed (r = 0.453, p < 0.01), but with small predictive value—coefficient of variation for regression was 45.5%; the average of the paired differences between the estimated and actual PMIs was 5.24 + 3.19 h. Although useful results might be expected due to the large transmembrane gradient for magnesium, the results of this study strongly disprove the usefulness of measuring magnesium in VH to estimate the time of death.     

Effect of Temperature on the Visualization by Digital Color Mapping of Latent Fingerprint Deposits on Metal

Visualization of fingerprint deposits by digital color mapping of light reflected from the surface of heated brass, copper, aluminum, and tin has been investigated using Adobe^® Photoshop^®. Metals were heated to a range of temperatures (T) between 50°C and 500°C in 50°C intervals with enhancement being optimal when the metals are heated to 250°C, 350°C, 50°C, and 300°C, respectively, and the hue values adjusted to 247°, 245°, 5°, and 34°, respectively. Fingerprint visualization after color mapping was not degraded by subsequent washing of the metals and color mapping did not compromise the visibility of the fingerprint for all values of T. The optimum value of T for fingerprint visibility is significantly dependent of the standard reduction potential of the metal with Kendall's Tau (τ) = 0.953 (p < 0.001). For brass, this correlation is obtained when considering the standard reduction potential of zinc rather than copper.     

Quantifying the Temperature of Maggot Masses and its Relationship to Decomposition

Numerous Calliphoridae species have been observed to form larval aggregations during the feeding stage of development, resulting in localized increases in temperature. This study investigates the relationship between maggot numbers in a mass and heat generation. Single‐species aggregations (Lucilia sericata) of various sizes (50–2500 individuals) were reared in the laboratory at a constant ambient temperature of 22°C. Internal mass temperatures were recorded every 5 min throughout the feeding stage of development. Results showed that mass temperatures increased with mass numbers (p‐value < 0.001), ranging from 2.5 to 14°C above ambient. A minimum mass size of 1200 produced overall temperatures that were significantly warmer than ambient, diverging away from 22°C after c. 26 h. These results indicate that the microclimate of a mass has the potential to differ significantly from ambient, which may be influencing larval development rates and should therefore be factored into mPMI estimates to increase accuracy.     

The Erratic Behavior of Lesions in Burnt Bone

This study analyses depressed fractures (by blunt force trauma) and circular full‐thickness injuries (drill injuries and gunshot wounds) in charred bones. Fifty bovine ribs (total 104 lesions) were divided into three groups. The first group consisted in 20 depressed hammer‐produced fractures; in the second one, 60 round drill‐holes were produced (30 circular, 30 semicircular); in the third group, 12 fleshed and 12 skeletonized ribs were hit by 9‐mm bullets. Each specimen was carbonized in an electric oven up to 800°C. Morphological and metric analyses were performed before and after: morphological features were preserved, but depressed fractures showed an increase in their dimensions (p‐value < 0.05); the drilled holes shrunk (p‐value < 0.01); the charring cycle increased the number of fractures in samples with gunshot wounds differently in fleshed and defleshed ribs. This study showed the complex behavior of charred bone, for what concerns the interpretation of trauma and how caution should be applied.     

Bacterial Degradation of Risperidone and Paliperidone in Decomposing Blood

The stability of two benzisoxazole antipsychotics was determined in vitro in decomposing porcine blood inoculated with bacteria, utilizing a high‐performance liquid chromatography with ultraviolet and fluorescence detection method for drug quantitation. Stability experiments for risperidone and paliperidone were conducted at 7, 20 and 37°C for 4 days using sterile and bacterially inoculated porcine blood. The drugs were stable in sterile blood at each temperature and in inoculated blood at 7°C, but degraded significantly in inoculated blood at 20 and 37°C. Complete loss occurred within 2 days when incubated at 37°C. The benzisoxazole‐cleaved degradation products for both drugs were identified as 2‐hydroxybenzoyl‐risperidone and 2‐hydroxybenzoyl‐paliperidone utilizing liquid chromatography quadrupole‐time‐of‐flight mass spectrometry and accurate mass measurements. The degradation products have been found in postmortem case studies, including one case where risperidone and paliperidone were not detected, indicating complete conversion can occur in situ.     

Variation in Developmental Time for Geographically Distinct Populations of the Common Green Bottle Fly,Lucilia sericata (Meigen)*

Abstract: Time between death and discovery of remains, or postmortem interval (PMI), can be assessed using blow fly maggot age. Forensic entomologists rely on published, often nonlocal, species‐specific developmental tables to determine maggot age. In a series of common garden experiments, we investigated the developmental rate variation between populations of Lucilia sericata collected from Sacramento, CA, San Diego, CA, and Easton, MA at 16°C, 26°C, and 36°C. For the 16°C trial the time measurement started at egg hatch, while for the higher temperatures the experiment began at oviposition; the wandering stage signified the endpoint for all experiments. The distribution of developmental times differed significantly (ANOVA, p < 0.001) between the three populations within each temperature treatment. We discovered that regional variation of developmental times within a blow fly species exists. This study demonstrates the importance of assembling local population‐specific developmental tables when estimating larval age to determine PMI.     

Brain Fluid Content Related to Body Position and Postmortem Interval – An Animal Model

Adult male Sprague Dawley rats were euthanized and placed in a horizontal or vertical (head‐down) position at room temperature, after which brain fluid content was measured by a moisture analysis technique at variable time points. No significant difference in brain fluid content was observed between horizontal and vertical postmortem positions. A significant increase in brain fluid content was demonstrated 3, 6, and 24 h after death, with maximal fluid content observed at 24 h. Specifically, the brain fluid content of control animals was 77.79 ± 0.36%, increasing to 80.05 ± 0.22% at 24 h (p < 0.0001). This study has demonstrated no significant differences in brain fluid content related to postmortem position, suggesting that a head‐down position is not associated with increased brain fluid content or swelling. However, significant temporal increases in brain fluid content after death, most likely related to cerebral liquefaction, occur.     

A Preliminary Examination of Differential Decomposition Patterns in Mass Graves

Five pairs of mass graves, each containing carcasses of 21 rabbits, were used to examine differential decomposition at four locations within the burial: surface, deep, mid‐outer, and core. Every 100 accumulated degree days (ADD), a pair of graves was exhumed, and total body score (TBS) and internal carcass temperature of each rabbit were recorded. Decomposition did not differ for core‐ and deep‐positioned carcasses (p = 0.13); differences were significant (p < 0.001) for all other location comparisons. Decomposition occurred fastest in shallow carcasses, followed by mid‐outer carcasses; deep and core carcasses decomposed slowest and at rates not significantly different from one another. Adipocere formation was minimal and confined to deep carcasses. Carcass location within the mass grave significantly influenced internal carcass temperature (p < 0.001); a mean internal temperature difference of ca. 1°C existed between deep and shallow carcasses (30 cm apart). Effects of mass compactness and oxygenation require further investigation.     

Radiodense bullet wipe around osseous entrance gunshot wounds

“Bullet wipe” is the material deposited by a bullet on any surface with which it comes into contact after it is fired and may contain debris from the gun barrel, including particles of primer and metal fragments from previously fired bullets. X‐ray analysis is a non‐destructive method by which traces of metallic elements can be visually detected. The analysis of osseous defects for radiodense bullet wipe (RBW) assists in determining the presence or absence of perforating gunshot wounds, especially in fragmented, skeletonized remains. The aim of our current study was to determine the frequency of RBW around entrance firearms injuries that perforated bone. We prospectively analyzed entrance gunshot wounds for RBW over a three‐year period using digital X‐ray analysis (n = 59). We retrospectively reviewed the corresponding autopsy reports to determine the frequency of RBW by biologic sex, reported ancestry, age‐at‐death, location of wound, manner of death, range of fire, bullet caliber, and presence of bullet jacket. Data were analyzed by Fisher's exact test or Chi‐square test with significance levels accepted at p < 0.05. RBW was present in 66% (n = 39) of examined cases. Decedent characteristics did not significantly alter RBW distribution, including biologic sex (p = 0.75), reported ancestry (p = 0.49), and age‐at‐death (p = 0.43). Additionally, the location of the osseous entrance gunshot wound, manner of death, range of fire, and cartridge caliber did not affect RBW detection. All cases involving non‐jacketed rounds (n = 5) showed RBW (p = 0.30). To our knowledge, this study is the first to report the frequency of RBW detection from osseous entrance gunshot wounds.     

Time- and Temperature-Dependent Changes in Cytochrome c Oxidase Activity and Cyanide Concentration in Excised Mice Organs and Mice Cadavers

Postmortem stability of cyanide biomarkers is often disputed. We assessed the time and temperature-dependent changes in cytochrome c oxidase (CCO) activity and cyanide concentration in various organs of mice succumbing to cyanide. Immediately after death, excised mice organs and mice cadavers were stored at room temperature (35°C ± 5°C) or in frozen storage (−20°C ± 2°C). At various times after death, CCO activity and cyanide concentrations were measured in excised mice organs or organs removed from mice cadavers. The study revealed that (i) measuring both the biomarkers in mice cadavers was more reliable compared to excised mice organs, (ii) measuring temporal CCO activity and cyanide concentration in vital organs from mice cadavers (room temperature) was reliable up to 24 h, and (iii) CCO activity in the brain and lungs and cyanide concentration in organs from mice cadavers (frozen) were measurable beyond 21 days. This study will be helpful in postmortem determination of cyanide poisoning.     

Influence of Muzzle Instability on Bullet Deflection after Perforating Laminated Particleboards

The influence of muzzle instability on bullet deflection, when perforating laminated particleboards, was studied with three different handgun bullet types. The mean deflection angles of .32 Auto FMJ and .38 Special SJHP bullets were calculated to be 0.90° and 0.83°, respectively after perforating particleboards orthogonally at a 1 m muzzle-to-target distance. The mean angles dropped to 0.70° and 0.58° at a 15 m muzzle-to-target distance. The differences in deflection angles proved to be statistically significant (p < 0.05) with p-values of 0.023 and 0.001, respectively. The mean calculated deflection angles of .38 Special LRN bullets also dropped from 1.51° to 1.38° when the muzzle-to-target distance was increased from 1 to 15 m, but this difference was not significant (p-value of 0.357). The results support the hypothesis that muzzle instability has an influence on deflection. The possible implications for shooting incident reconstructions and for future research are discussed.     

A Checklist of Calliphoridae Blowflies (Insecta,Diptera) Associated with a Pig Carrion in Central Brazil*

Abstract: Although the Cerrado is the second major Brazilian biome, few studies have been undertaken about its entomofauna. Blowflies have an important role in forensic entomology, helping in the determination of postmortem intervals. The main goal of this exploratory study was to identify and to catalog the blowfly species associated with a pig carcass. The study was conducted in a pasture in Brasília, Distrito Federal. A pig (Sus scrofa) was killed with a .22 caliber shot in the frontal region of the head. Adult blowflies were surveyed daily from June 1 through 30, 2004. A total of 14,910 adult calliphorids were collected, representing eight species: Chrysomya albiceps, C. megacephala, Cochliomyia macellaria, Chloroprocta idioidea, Hemilucilia semidiaphana, H. segmentaria, Lucilia cuprina, and L. eximia. C. albiceps was the most frequent species, amounting to 94.76% of the catch. Five decomposition stages were observed, and for calliphorids, the most attractive stage was the bloated one.     

Can Neck Contusions in Unexplained Pediatric Deaths be Explained by Cardiopulmonary Resuscitation?

The distribution of cutaneous contusions in infants may raise the possibility of maltreatment. Neck contusions are particularly problematic since they seldom occur outside the setting of abuse, while cardiopulmonary resuscitation (CPR)‐related maneuvers may involve the neck. To address the role of CPR in neck findings, we examined 260 consecutive pediatric autopsies in which CPR was attempted. No neck contusions were identified in manners of death classified as natural, undetermined, or suicide. Contusions were present in two of 80 deaths classified as accident and had obvious accidental causes. About 26% of deaths classified as homicide had neck contusions with no explanation provided by the caregiver (p < 0.0001). In conclusion, neck contusions in deceased children with no apparent explanation should be regarded as suspicious for abuse and investigated accordingly. CPR is not a plausible explanation for neck contusions in children.     

Investigations into Enhancing Yersinia pestis Cells Viability following Environmental Sampling for Forensic Analysis

Following an intentional or accidental bio-warfare agent (BWA) release, environmental sample analysis is absolutely critical to determine the extent of contamination. When dealing with nonspore forming BWA (e.g., Yersinia pestis), retention of cell viability is central to such analyses. Even though significant advances have been achieved in DNA sequencing technologies, a positive identification of BWAs in environmental samples must be made through the ability of cells to form colony-forming units upon culturing. Inability to revive the cells between collection and analysis renders such studies inconclusive. Commercial kits designed to preserve the viability of pathogens contained within clinical samples are available, but many of them have not been examined for their ability to preserve samples containing suspected BWAs. The study was initiated to examine the applicability of commercial solutions aiding in retention of Y. pestis viability in samples stored under nonpermissive temperatures, that is, 40 and 37°C. While none of the tested solutions sustained cell viability at 40°C, the results show five out of 17 tested preservatives were capable of supporting viability of Y. pestis at 37°C.     

An Examination of the Transition of Fracture Characteristics in Long Bones from Fresh to Dry in Central Florida: Evaluating the Timing of Injury

It is important to conduct timing of injury research analyzing fracture characteristics at known postmortem intervals (PMI) because bone can retain fresh characteristics throughout the PMI. Defleshed pig (Sus scrofa) long bones were fractured weekly in two environments (full sun and shade) over 14 weeks in Central Florida and fracture characteristics were categorized (N = 136) for analysis. Results of analysis of variance (ANOVA) using time in weeks (PMI) as a dependent variable indicate significant relationships between PMI and Fracture Angle (p < 0.001), Fracture Surface (p < 0.001), and Fracture Outline (p < 0.001). Fracture characteristics associated with perimortem trauma (smooth Fracture Surfaces and curved or V‐shaped Fracture Outlines) were commonly observed. Analysis of fracture characteristics for each environment demonstrated similar patterns. Overall, the loss of only fresh fracture characteristics for each bone was noted earlier in the PMI for the Central Florida region than previously reported.     

Scanning Electron Microscopy–Energy‐Dispersive X‐Ray (SEM/EDX): A Rapid Diagnostic Tool to Aid the Identification of Burnt Bone and Contested Cremains

This study investigates the use of Scanning electron microscopy–energy‐dispersive X‐ray (SEM‐EDX) as a diagnostic tool for the determination of the osseous origin of samples subjected to different temperatures. Sheep (Ovis aries) ribs of two experimental groups (fleshed and defleshed) were burned at temperatures of between 100°C and 1100°C in 100°C increments and subsequently analyzed with the SEM‐EDX to determine the atomic percentage of present elements. Three‐factor ANOVA analysis showed that neither the exposure temperature, nor whether the burning occurred with or without soft tissue present had any significant influence on the bone's overall elemental makeup (p > 0.05). The Ca/P ratio remained in the osseous typical range of between 1.6 and 2.58 in all analyzed samples. This demonstrates that even faced with high temperatures, the overall gross elemental content and atomic percentage of elements in bone remain stable, creating a unique “fingerprint” for osseous material, even after exposure to extreme conditions.     

Calculating Point of Origin of Blood Spatter Using Laser Scanning Technology

The point of origin of an impact pattern is important in establishing the chain of events in a bloodletting incident. In this study, the accuracy and reproducibility of the point of origin estimation using the FARO Scene software with the FARO Focus^3D laser scanner was determined. Five impact patterns were created for each of three combinations of distances from the floor (z) and the front wall (x). Fifteen spatters were created using a custom impact rig, scanned using the laser scanner, photographed using a DSLR camera, and processed using the Scene software. Overall results gave a SD = 3.49 cm (p < 0.0001) in the x‐direction, SD = 1.14 cm (p = 0.9291) in the y‐direction, and SD = 9.08 cm (p < 0.0115) in the z‐direction. The technique performs within literature ranges of accepted accuracy and reproducibility and is comparable to results reported for other virtual stringing software.