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.     

Validation of chest X-ray comparisons for unknown decedent identification

Comparing skeletal structures between antemortem and postmortem chest radiographs is widely used by forensic specialists from many disciplines to positively identify unknown decedents. However, validity assessments of this method have been fairly limited. This study had three objectives: 1) to quantify the reliability of ante- and postmortem chest radiograph comparison for decedent identification; 2) to identify useful radiologic features supporting decedent identification; and 3) to recognize sources of error in decedent identification related to use of comparative radiographs. A forensic pathologist, a forensic anthropologist, and two radiologists participated in the study. Our results showed that chest radiograph comparisons proved reliable, if basic decedent information was provided, and antemortem and postmortem radiographs were adequately positioned and exposed. A "morphological approach" using normal anatomical structures for comparison may provide the most efficient method for accurate identification.     

Validation of a 16-locus fluorescent multiplex system

STR multiplexes have been indispensable for the efficient genotyping of forensic samples. The PowerPlex 16 System contains the coreCODIS loci, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, THOI, TPOX, vWA, the sex determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. This multiplex satisfies the locus requirements for most national databases and is the most efficient currently available system due to its single PCR amplification. To provide the groundwork for judicial acceptance, including the publication of primer sequences, and to evaluate laboratory-to-laboratory variation, a developmental validation for casework on this commercially available system was performed in 24 laboratories and produced the following conclusions. Amplification was reliable on a variety of thermal cyclers and product could be analyzed on either an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer. Genotyping using single source samples was consistent between 0.25 and 2 ng of input DNA template with a few laboratories obtaining complete genotypes at 0.0625 ng. However, heterozygote allele imbalance (<60% peak height balance) caused by stochastic effects was observed at a rate of 13% with 0.125 ng DNA and 22% at 0.0625 ng DNA. Mixture analyses were done using a total of 1 ng of DNA template. Most alleles were detected in mixtures of 4 to 1 and some minor alleles were detected in mixtures of 19 to 1. Optimum amplification cycle number was dependent on the sensitivity of the detection instrument used and could also be adjusted to accommodate larger amounts of DNA on solid supports such as FTA paper. Reaction conditions including volume, annealing temperature, and concentrations of primer, AmpliTaq Gold, and magnesium were shown to be optimal yet robust enough to withstand moderate variations without affecting genotype analysis. Environmental, matrix and standard source analyses revealed an ability to obtain complete genotypes in all sample types except those exposed to 80 degrees C for 12-48 days. Finally, comparison of genotype results from the PowerPlex 16 System with other commercially available systems on non-probative reference and forensic samples showed consistent results.     

An improved method for post-PCR purification for mtDNA sequence analysis

Mitochondrial DNA (mtDNA) analysis of forensic samples typically is performed when the quantity and quality of DNA are insufficient for nuclear DNA analysis or when maternal relatives may be the only reference source. Many of the steps required in the analytical process are both lengthy and labor intensive. Therefore, improvements in the process that reduce labor without compromising the quality of the data are desirable. The current procedure requires purification of the amplicons by centrifugal filtration after PCR and prior to cycle sequencing. Because this method requires several manipulations to perform, alternate cleanup procedures were investigated. These include the use of 1) Qiagen QlAquick PCR Purification columns, 2) Concert Rapid PCR Purification columns, and 3) ExoSAP-IT reagent. When the yield of purified amplicons, quality of the sequence profile, and ease of assay were evaluated, the use of ExoSAP-IT reagent for post-amplification purification was chosen to replace the filtration method.     

Role of alcohol in heroin overdose

The relationship between ethanol and risk of heroin overdosage was studied. Statistical processing of the results of forensic chemical analysis (460 expert evaluations) carried out in Chelyabinsk Regional Bureau of Forensic Medical Expert Evaluations in 2000 was carried out. The results of morphine and ethanol measurements in the blood and urine from corpses where deaths ensued from narcotic or ethanol poisoning, were analyzed. The concentrations of morphine in the blood and urine were measured on a gaseous chromatographer with mass-selective detector (Hewlett Packard HP 6890/HP-5972). Methods for measuring urinary and blood morphine are described. The results of statistical analysis demonstrated relationships between the age and ethanol concentrations in the blood and urine; blood ethanol and total urinary and blood morphine concentrations; blood concentration of free morphine and presence of 6-monoacetylmorphine in the blood. The authors conclude that the presence of ethanol in the blood together with morphine drastically augments the risk of rapid death from respiration arrest. It can also lead to a relatively high risk of overdosage in experienced narcomaniacs using heroin and ethanol.     

Crystallization of cerebrospinal fluid in cases of death from ischemic heart disease and ethanol poisoning

Crystallographic analysis of the cerebrospinal fluid (CSF) was carried out in 18 cases of death from coronary disease and 19 cases of death from ethanol poisoning. The crystallograms were evaluated visually and by the stereoscopic picture. Specific features of the CSF crystal colonies growth in subjects dead from the above conditions are determined.