Quantitative analysis of amplifiable DNA in tissues exposed to various environments using competitive PCR assays

Competitive PCR assays were established for the mitochondrial DNA hypervariable region I and the human amelogenin locus. Using these assays, the copy numbers of DNA participating in PCR (amplifiable DNA) were quantified in tissues exposed to different environments. Human ribs, skin and nails were left in three exposure conditions (in the open air, in soil and in water). The amounts of amplifiable DNA in these tissues were quantified during a time period of up to two months. The amount of amplifiable DNA was well preserved in hard tissues (ribs and nails) regardless of the exposure conditions, whereas the soft tissues immersed in water showed a rapid decrease in amplifiable DNA. Strong PCR inhibition was observed in the DNA extracts obtained from buried bones. This phenomenon was clearly identified from an amplification failure of the internal standards in the competitive PCR. A preliminary examination to identify the PCR inhibitor suggested that the soil itself contributed to the inhibition. In addition, the amounts of amplifiable DNA in case samples were also investigated.     

An analysis of factors contributing to a series of deaths caused by exposure to high environmental temperatures

Autopsy reports at the Forensic Science Centre, Adelaide, South Australia, were reviewed for the 8 years from January 1991 to December 1998 for cases with unusual features in which deaths had been attributed to exposure to high environmental temperatures. Amphetamine-related hyperpyrexial deaths, anesthetic deaths caused by malignant hyperpyrexia, deaths of elderly incapacitated individuals during heat waves, and deaths of children trapped in the back of cars were excluded from the study. In 9 cases, where heat-related deaths had occurred (age range 21 to 77 years; M:F = 8:1). Predisposing factors included lack of familiarity with Australian environmental conditions, excessive clothing, prolonged sun exposure, acute alcohol intoxication, obesity, benztropine and trifluoperazine medication, and underlying dementia, alcoholic liver disease, and possibly epilepsy.     

Detection of methamphetamine in mouse femurs exposed to high temperature

Bone samples are valuable for examining the cause of death and circumstance leading up to death when body fluids are not available for forensic toxicological analysis. Examined were heat-induced changes in methamphetamine and amphetamine concentrations in femurs removed from methamphetamine-injected mice to determine if the burned bones could be used for toxicology testing. The femurs were heated at 100°C, 300°C, or 500°C for 10 or 30 min. The tissue structure of the heated femurs was preserved at 100°C for 30 min but was destructed at higher temperatures. Methamphetamine and amphetamine were detected in femurs heated at 100°C for 10 min, 100°C for 30 min, and 300°C for 10 min (with methamphetamine and amphetamine concentrations ranging from 0.36 to 35 μg/g and 0.54 to 47 μg/g, respectively). Methamphetamine and amphetamine were detectable when heated above their decomposition temperature as a result of limited heat transfer do to protection provide by the femoral muscle. Thus, the bone could be a useful analytical sample in cases of burn-related deaths, where it is difficult to collect body fluids.     

Study about the effect of high temperatures on STRs typing

Forensic investigations generally involve samples that have unknown storage conditions.These conditions may help to speed up or slow down the degradation of DNA. For example environmental factors that speed up the decay include: UV light, humidity, and temperature. The aim of the present study is to evaluate the effect of high temperature on the ability to perform DNA extraction and typing from different biological fluids (blood, saliva, and semen) after 20 min incubation in an oven at different temperatures 50 °C, 100 °C, 150 °C, and 200 °C or direct exposition to the effect of a flame for few minutes. Our results support the ability to type DNA even from samples exposed to drastic condition.     

Radiographic evaluation of teeth subjected to high temperatures: experimental study to aid identification processes

The radiographic evaluation of dental remains represents a significant aspect in the forensic identification process, particularly after an exposure to fire. The aim of this "in vitro" study was to evaluate the radiographic features of unrestored, endodontically treated and restored teeth after exposure to an experimental range of high temperatures. Ninety human teeth were divided into two groups: (1) unrestored teeth, as a control group and (2) teeth endodontically treated (condensation technique) and restored with amalgam or composite fillings. Before testing the high temperatures, periapical radiographs of all teeth were performed. The tests of exposure to heat were carried out in an oven for six different temperatures (200, 400, 600, 800, 1000 and 1100 degrees C (392, 752, 1112, 1472, 1832, 2012 degrees F)). After each exposure, periapical radiographs of all the teeth were taken. The radiographic appearance of all the teeth before and after the thermal stresses were evaluated and the differences were recorded. The results of the radiographic examination showed that a number of significant radiographic details were conserved: the composite fillings were in place maintaining the shape till 600 degrees C (1112 degrees F), the amalgam fillings were in place maintaining the shape till 1000 degrees C (1832 degrees F) and the endodontic treatments were recognisable till 1100 degrees C (2012 degrees F).     

Changes in the concentration of carboxyhemoglobin in the blood under the action of high temperatures

Changes in carboxyhemoglobin concentrations are studied in 18 specimens of cadaveric blood 1-2 h and 3 days after heating to 50, 65, 70, 75, 80, and 90 degrees C for 5, 10, and 20 min. The concentration of carboxyhemoglobin decreased to 15% after heating at 70-75 degrees C for 10 and 20 min. Heating to 80-90 degrees makes the measurements impossible. Five-min heating at 50-65 degrees C did not change the concentration of carboxyhemoglobin in the blood.     

Three cases of sudden death due to butane or propane gas inhalation: analysis of tissues for gas components

We report three cases of sudden death due to inhalation of portable cooking stove fuel (case 1), cigarette lighter fuel (case 2), and liquefied petroleum gas (LPG) (case 3). Specimens of blood, urine, stomach contents, brain, heart, lung, liver, kidney, and fat were collected and analyzed for propylene, propane, isobutane, and n-butane by headspace gas chromatography. n-Butane was the major substance among the volatiles found in the tissues of cases 1 and 2, and propane was the major substance in case 3. A combination of the autopsy findings and the gas analysis results revealed that the cause of death was ventricular fibrillation induced by hard muscle exercise after gas inhalation in cases 1 and 2, and that the cause of death in case 3 might be hypoxia. It is possible that the victim in case 3 was under anesthetic toxicity of accumulated isobutane which is a minor component of liquefied petroleum gas.     

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

New formulae for estimating age-at-death in the Balkans utilizing Lamendin's dental technique and Bayesian analysis

The present study analyzed apical translucency and periodontal recession on single-rooted teeth in order to generate age-at-death estimations using two inverse calibration methods and one Bayesian method. The three age estimates were compared to highlight inherent problems with the inverse calibration methods. The results showed that the Bayesian analysis reduced severity of several problems associated with adult skeletal age-at-death estimations. The Bayesian estimates produced a lower overall mean error, a higher correlation with actual age, reduced aging bias, reduced age mimicry, and reduced the age ranges associated with the most probable age as compared to the inverse calibration methods for this sample. This research concluded that periodontal recession cannot be used as a univariate age indicator, due to its low correlation with chronological age. Apical translucency yielded a high correlation with chronological age and was concluded to be an important age indicator. The Bayesian approach offered the most appropriate statistical analysis for the estimation of age-at-death with the current sample.     

HPLC simultaneous determination of glycerol and mannitol in human tissues for forensic analysis

A method for simultaneous determination of glycerol and mannitol in various human tissues was devised and for this we used high-performance liquid chromatography (HPLC). Specimens were homogenized in a mixture of chloroform and methanol, phosphate buffer (pH 7.0) and pentaerythritol (IS) solution. After centrifugation, an aliquot of the aqueous layer was evaporated to dryness and derivatized with p-nitrobenzoyl chloride at 50 degrees C for 1h, then applied to HPLC with analytical conditions of: column, CAPCELL PAK C18 MG (250 mm x 3.0 mm i.d., 5 microm, Shiseido Co. Ltd., Tokyo, Japan); column temperature, 1-2 degrees C; mobile phase, 75% acetonitrile-distilled water containing 0.05% trifluoroacetic acid, 0.05% heptafluoro-n-butyric acid and 0.1% triethylamine; flow rate, 0.5 ml/min; wavelength, 260 nm. Calibration curves for both substances were linear in concentration ranges from 1 to 500 microg/0.1g and correlation coefficients exceeded 0.99. The relative standard deviation (R.S.D.) of the method was evaluated at concentrations of 10 and 100 microg/0.1g, and ranged from 0.84 to 10.6%. Using this method, we determined the regional distribution levels of glycerol and mannitol in various tissues from an autopsied brain dead man.     

A comparison of three shoe sole impression lifting methods at high substrate temperatures

Footwear impressions are a common form of evidence found at crime scenes, and the accurate recovery and recording of such impressions is critical for shoe sole comparison and identification. The lifting of shoe sole impressions from hot surfaces (>30°C/86°F) and in hot environments has received little attention in the literature, particularly in relation to the recovery of class and randomly acquired characteristics (RACs) required for accurate comparisons. This study addressed this knowledge gap by comparing the performance of three common impression lifters (gelatin, adhesive, and vinyl static cling film) at recovering shoe sole impressions in dust from hot flooring substrates. Dry origin dust shoe sole impressions were made on ceramic tile, galvanized metal, and laminated wood flooring using a shoe that possessed two RACs and five class characteristics present on the sole. Substrates were left in direct full sun for five hours during a summer day prior to lifting. Performance was measured by the proportion of RACs and class characteristics visible in each lifted impression. Results demonstrated that the vinyl static cling film tested performed poorly across all substrates, particularly for metal (23.8% marks recovered), including notable shrinkage of the lifted impression. In contrast, adhesive (~96% marks recovered over all substrates), and to a lesser extent gelatin (~85%), lifts were highly successful on hot substrates. These data suggest that adhesive lifts can consistently and accurately recover shoe sole impressions from hot substrates. This study contributes critical information for crime scene examiners to improve and expand evidence recovery in hot environments.     

Reactions of latent prints exposed to blood

We explored whether an undeveloped latent print (fingermark) exposed to blood and later developed by enhancement with blood reagents such as amido black (AB) or leucocrystal violet (LCV) could appear as a genuine blood mark. We examined three different experimental conditions. In Experiment I, fingermark residue only was tested, as a control to confirm that fingermark residue alone does not react with the blood reagents AB and LCV. Experiment II investigated whether latent fingermarks exposed to blood dilutions could be treated with AB or LCV and subsequently appear as a genuine blood mark enhanced with AB or LCV. Experiment III tested whether latent fingermarks exposed to whole blood could be processed with AB or LCV and subsequently appear as a genuine blood mark enhanced with AB or LCV.The present study found that indeed, fingermark residue alone does not react with the blood reagents AB and LCV. In Experiment II, an interaction occurred between the fingermark residue and the diluted blood that caused the ridges to appear a red color. In the present study, this interaction is called a faux blood mark. While the faux blood mark phenomenon occurred most often following exposure to diluted blood, it did not occur consistently, and a predictable pattern could not be established. However, the reaction occurred more frequently following extended fingermark residue drying times. Faux blood marks are distinguishable from genuine blood marks prior to enhancement with blood reagents. Following treatment with blood reagents, it became increasingly difficult to determine whether the enhanced mark was a genuine blood print or a latent fingermark exposed to diluted blood. Latent fingermarks exposed to whole blood often resulted in a void prior to enhancement, but following treatment with blood reagents, were difficult to distinguish from a genuine blood mark enhanced with blood reagents.     

Computer-based production of comparison overlays from 3D-scanned dental casts for bite mark analysis

Bite mark analysis assumes the uniqueness of the dentition can be accurately recorded on skin or an object. However, biting is a dynamic procedure involving three moving systems, the maxilla, the mandible, and the victim's reaction. Moreover, bite marks can be distorted by the anatomic location of the injury or the elasticity of the skin tissue. Therefore, the same dentition can produce bite marks that exhibit variations in appearance. The complexity of this source of evidence emphasizes the need for new 3D imaging technologies in bite mark analysis. This article presents a new software package, DentalPrint (2004, University of Granada, Department of Forensic Medicine and Forensic Odontology, Granada, Spain) that generates different comparison overlays from 3D dental cast images depending on the pressure of the bite or the distortion caused by victim-biter interaction. The procedure for generating comparison overlays is entirely automatic, thus avoiding observer bias. Moreover, the software presented here makes it impossible for third parties to manipulate or alter the 3D images, making DentalPrint suitable for bite mark analyses to be used in court proceedings.     

Detection of metals in human tissues by spectrum analysis

Potentialities of spectrography experts were demonstrated to be helpful to the employees of patient-care facilities (expertise and research institutions) in solving a variety of burning issues by joint effort. Tissue samplings, hairs and daily urine were comparatively analyzed, by the example of using the low-friction hip arthroplasty by comochrome friction Protasu-21WF, for the content of microelements (ME) belonging to the hip endoprosthesis. We found that MEs (Co, Cr, Mo) are mostly discharged during the first year of endoprosthesis utilization, after which, a regular reduction of concentrations was noted. No exceeding norms were registered in any cases, which denotes a minimal wear of the friction pair.     

Effects of individual dental factors on genomic DNA analysis

The use in forensic medicine of methods pertaining to molecular biology has made it possible to identify human remains through the analysis of polymorphic profiles of human DNA. Voluntary, accidental, or natural postmortem degradation, as well as environmental conditions, influences the preservation state of the corpse, making it sometimes difficult to obtain biologic material suitable for genetic analysis (e.g., hair, soft and/or hard tissue). According to their anatomic/morphologic characteristics, dental formations are particularly resistant to external insults and are thus suitable for this kind of research. The purpose of this work, conducted on nonselected dental findings (presenting intrinsic characteristics similar to those usually found in forensic cases) that were homogeneous with regard to environmental factors, was to determine an operative protocol that will enable combination of the maximum availability of genomic DNA with the preservation of the morphologic characteristics of the tooth for classic anthropologic evaluations.     

人离体肝组织胺类产生与死亡时间和环境温度的关系

目的探讨在不同环境温度下,人离体肝组织中有机胺类产生量随死亡时间变化的规律,为某些碎尸案的死亡时间推断提供参考资料。方法应用高效液相色谱仪外标法,动态检测人离体肝组织中组胺、腐胺、尸胺和未腐败氨基酸含量。结果在8℃、15℃、23℃和32℃环境下,有机胺类产生量分别于人死后肝脏即刻离体69h、54h、40h和33h趋于最大值,并且在到达峰值之前,各温度组离体肝组织中有机胺类生成量均与死亡时间成正比关系。结论人离体肝组织中有机胺类产生量与死亡时间和环境温度在一定范围内均呈正相关。     

The use of SEM/EDS analysis to distinguish dental and osseus tissue from other materials

With increasing frequency, relatively small, fragmentary evidence thought to be osseous or dental tissue of human origin is submitted to the forensic laboratory for DNA analysis with the request for positive identification. Prior to performing DNA analysis, however, it is prudent to first perform a presumptive test or "screen" to determine whether the questioned material may be eliminated from further consideration. When material is shown not to be consistent with bone/teeth, DNA testing is not performed. When such determinations cannot be made from gross morphological features, elemental analysis can be indicative. This presumptive test is made possible by applying scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) in conjunction with an X-ray spectral database recently developed by the FBI laboratory. This database includes spectra for many different materials including known examples of bone and tooth from many different contexts and representing the full range of taphonomic conditions. Results of SEM/EDS analysis of evidence can be compared to these standards to determine if they are consistent with bone and/or tooth and, if not, then what the material might represent. Analysis suggests that although the proportions and amounts of calcium and phosphorus are particularly important in differentiating bone and tooth from other materials, other minor differences in spectral profile can also provide significant discrimination. Analysis enables bone and tooth to be successfully distinguished from other materials in most cases. Exceptions appear to be ivory, mineral apatite, and perhaps some types of corals.