Development of a dynamic headspace concentration technique for the non-contact sampling of human odor samples and the creation of canine training aids

Human scent and human remains detection canines are used to locate living or deceased humans under many circumstances. Human scent canines locate individual humans on the basis of their unique scent profile, while human remains detection canines locate the general scent of decomposing human remains. Scent evidence is often collected by law enforcement agencies using a Scent Transfer Unit, a dynamic headspace concentration device. The goals of this research were to evaluate the STU-100 for the collection of human scent samples, and to apply this method to the collection of living and deceased human samples, and to the creation of canine training aids. The airflow rate and collection material used with the STU-100 were evaluated using a novel scent delivery method. Controlled Odor Mimic Permeation Systems were created containing representative standard compounds delivered at known rates, improving the reproducibility of optimization experiments. Flow rates and collection materials were compared. Higher air flow rates usually yielded significantly less total volatile compounds due to compound breakthrough through the collection material. Collection from polymer and cellulose-based materials demonstrated that the molecular backbone of the material is a factor in the trapping and releasing of compounds. The weave of the material also affects compound collection, as those materials with a tighter weave demonstrated enhanced collection efficiencies. Using the optimized method, volatiles were efficiently collected from living and deceased humans. Replicates of the living human samples showed good reproducibility; however, the odor profiles from individuals were not always distinguishable from one another. Analysis of the human remains samples revealed similarity in the type and ratio of compounds. Two types of prototype training aids were developed utilizing combinations of pure compounds as well as volatiles from actual human samples concentrated onto sorbents, which were subsequently used in field tests. The pseudo scent aids had moderate success in field tests, and the Odor pad aids had significant success. This research demonstrates that the STU-100 is a valuable tool for dog handlers and as a field instrument; however, modifications are warranted in order to improve its performance as a method for instrumental detection.

The application of reduced-size short tandem repeat primer sets in the analysis of human DNA from degraded and compromised samples

The purpose of this research was to demonstrate the applicability of reduced-size STR (Miniplex) primer sets to challenging samples and to provide the forensic community with new information regarding the analysis of degraded and inhibited DNA. The Miniplex primer sets were validated in accordance with guidelines set forth by the Scientific Working Group on DNA Analysis Methods (SWGDAM) in order to demonstrate the scientific validity of the kits. The Miniplex sets were also used in the analysis of DNA extracted from human skeletal remains and telogen hair. In addition, a method for evaluating the mechanism of PCR inhibition was developed using qPCR. The Miniplexes were demonstrated to be a robust and sensitive tool for the analysis of DNA with as low as 100 pg of template DNA. They also proved to be better than commercial kits in the analysis of DNA from human skeletal remains, with 64% of samples tested producing full profiles, compared to 16% for a commercial kit. The Miniplexes also produced amplification of nuclear DNA from human telogen hairs, with partial profiles obtained from as low as 60 pg of template DNA. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for forensic analysis of degraded DNA from human skeletal remains, telogen hairs, and other challenging samples. In the evaluation of inhibition by qPCR, the effect of amplicon length and primer melting temperature was evaluated in order to determine the binding mechanisms of different PCR inhibitors. Several mechanisms were indicated by the inhibitors tested, including binding of the polymerase, binding to the DNA, and effects on the processivity of the polymerase during primer extension. The data obtained from qPCR illustrated a method by which the type of inhibitor could be inferred in forensic samples, and some methods of reducing inhibition for specific inhibitors were demonstrated. An understanding of the mechanism of the inhibitors found in forensic samples will allow analysts to select the proper methods for inhibition removal or the type of analysis that can be performed, and will increase the information that can be obtained from inhibited samples.

The use of quantitative polymerase chain reaction amplification method to determine sex ratio of human spermatozoa in semen and to determine maternal contamination of amniotic fluid samples

We have modified a technique which uses a single pair of primer sets directed against homologous but distinct genes on the X and Y chromosomes, all of which are coamplified in the same reaction tube with trace amounts of radioactivity. The resulting bands are equal in length, yet distinguishable by restriction enzyme sites generating two independent bands, a 364 bp X-specific band and a 280 bp Y-specific band. A standard curve was generated to show the linear relationship between X/Y ratio average vs. %Y or %X chromosomal content. Of the 51 purified amniocyte DNA samples analyzed, 16 samples showed evidence of high % X contamination while 2 samples demonstrated higher % Y than the expected 50% X and 50% Y chromosomal content. With regards to the 25 processed sperm samples analyzed, X-sperm enrichment was evident when compared to the primary sex ratio whereas Y-sperm was enriched when we compared before and after selection samples.

The analytical determination of the uniqueness and persistence of the volatile components of human scent using optimized collection methods

The Locard exchange principle proposes that a person can not enter or leave an area or come in contact with an object, without an exchange of materials. In the case of scent evidence, the suspect leaves his scent in the location of the crime scene itself or on objects found therein. Human scent evidence collected from a crime scene can be evaluated through the use of specially trained canines to determine an association between the evidence and a suspect. To date, there has been limited research as to the volatile organic compounds (VOCs) which comprise human odor and their usefulness in distinguishing among individuals. For the purposes of this research, human scent is defined as the most abundant volatile organic compounds present in the headspace above collected odor samples. ^ An instrumental method has been created for the analysis of the VOCs present in human scent, and has been utilized for the optimization of materials used for the collection and storage of human scent evidence. This research project has identified the volatile organic compounds present in the headspace above collected scent samples from different individuals and various regions of the body, with the primary focus involving the armpit area and the palms of the hands. Human scent from the armpit area and palms of an individual sampled over time shows lower variation in the relative peak area ratio of the common compounds present than what is seen across a population. A comparison of the compounds present in human odor for an individual over time, and across a population has been conducted and demonstrates that it is possible to instrumentally differentiate individuals based on the volatile organic compounds above collected odor samples. ^

Assessing the sources, quantities, and trends on human waste contamination along protected south Florida ecosystem: A chemical tracer based study

The common occurrence of human derived contaminants like pharmaceuticals, steroids and hormones in surface waters has raised the awareness of the role played by the release of treated or untreated sewage in the water quality along sensitive coastal ecosystems. South Florida is home to many important protected environments ranging from wetlands to coral reefs which are in close proximity to large metropolitan cities. Since large portions of South Florida and most of the Florida Keys population are not served by modern sewage treatment plants and rely heavily on the use of inefficient septic systems; a comprehensive survey of selected human waste contamination markers is needed in these areas to assess water quality with respect to non-traditional micro-constituents. ^ This study reports the development and application of new sensitive and selective analytical methods for the fast screening of multiple wastewater tracers, classified as Emergent Pollutants of Concern (EPOC). Novel methods for the trace analysis of non-traditional markers of human-specific contamination such as aminopropanone were developed and used to assess the potential of non-traditional markers as wastewater tracers. ^ During our investigation, surface water samples collected from near shore environments along the South Florida were analyzed for fifteen hormones and steroids, and five commonly detected pharmaceuticals. The compounds most frequently detected were: coprostanol, cholesterol, estrone, β-estradiol, caffeine, triclosan and DEET. Concentrations of caffeine, bisphenol A and DEET were usually higher and more prevalent than the hormonal steroids. In general, it was found that common pharmaceuticals and steroids are widely present in major coastal environments in South Florida. It is also evident that aquatic bodies in heavily urbanized sectors such as the Miami River and Key Largo Harbor contain higher concentrations of several compounds while relatively open bay waters and agricultural areas show reduced chemical signatures. Concentrations of hormones in the Little Venice area of Marathon Key were above the Lowest Observable Effect Levels (LOELs) for several species, indicating that biological resources in this area are at risk. Water quality issues in some of these coastal water environments go beyond eutrophication, thus EPOC should be the target goal for future mitigation projects. ^

Variables affecting the collection and preservation of human scent components through instrumental and biological evaluations

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. ^ The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. ^ Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. ^ Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines. ^

Evaluation of contact and non-contact trapping efficiencies of human scent chemical profiles and their stabilities under different environmental conditions

Establishing an association between the scent a perpetrator left at a crime scene to the odor of the suspect of that crime is the basis for the use of human scent identification evidence in a court of law. Law enforcement agencies gather evidence through the collection of scent from the objects that a perpetrator may have handled during the execution of the criminal act. The collected scent evidence is consequently presented to the canines for identification line-up procedures with the apprehended suspects. Presently, canine scent identification is admitted as expert witness testimony, however, the accurate behavior of the dogs and the scent collection methods used are often challenged by the court system. The primary focus of this research project entailed an evaluation of contact and non-contact scent collection techniques with an emphasis on the optimization of collection materials of different fiber chemistries to evaluate the chemical odor profiles obtained using varying environment conditions to provide a better scientific understanding of human scent as a discriminative tool in the identification of suspects. The collection of hand odor from female and male subjects through both contact and non-contact sampling approaches yielded new insights into the types of VOCs collected when different materials are utilized, which had never been instrumentally performed. Furthermore, the collected scent mass was shown to be obtained in the highest amounts for both gender hand odor samples on cotton sorbent materials. Compared to non-contact sampling, the contact sampling methods yielded a higher number of volatiles, an enhancement of up to 3 times, as well as a higher scent mass than non-contact methods by more than an order of magnitude. The evaluation of the STU-100 as a non-contact methodology highlighted strong instrumental drawbacks that need to be targeted for enhanced scientific validation of current field practices. These results demonstrated that an individual's human scent components vary considerably depending on the method used to collect scent from the same body region. This study demonstrated the importance of collection medium selection as well as the collection method employed in providing a reproducible human scent sample that can be used to differentiate individuals.

The Effect of Sample and Sample Matrix on DNA Processing: Mechanisms for the Detection and Management of Inhibition in Forensic Samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

Characterization of engineered human lung tissue

Characterizing engineered human lung tissue is an important step in developing a functional tissue replacement for lung tissue repair and in vitro analysis. Small tissue constructs were grown by seeding IMR-90 fetal lung fibroblasts and adult microvascular endothelial cells onto a Polyglycolic acid (PGA) polymer template. Introducing the constructs to dynamic culture conditions inside a bioreactor facilitated three-dimensional growth seen in scanning electron microscopy images (SEM). Characterization of the resultant tissue samples was done using SEM imagery, tensile tests, and biochemical assays to quantify extra-cellular matrix (ECM) composition. Tensile tests of the engineered samples indicated an increase in the mechanical properties when compared with blank constructs. Elastin and collagen content was found to average 3.19% and 15.49% respectively in relation to total mass of the tissue samples. The presence of elastin and collagen within the constructs most likely explains the mechanical differences that we noted. These findings suggest that the necessary ECM can be established in engineered tissue constructs and that optimization of this procedure has the capacity to generate the load bearing elements required for construction of a functional lung tissue equivalent.

Variables Affecting the Collection and Preservation of Human Scent Components through Instrumental and Biological Evaluations

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines.

The effect of sample and sample matrix on DNA processing: Mechanisms for the detection and management of inhibition in forensic samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.^