Y-chromosome testing has become more prevalent in recent years as a means of identifying forensic samples using STRs or identifying biomarkers for disease or determining geographic origins of populations. Additionally, Y-chromosome analysis is especially useful in paternity testing as the Y chromosome is inherited paternally and the male-specific region of the Y chromosome does not undergo any recombination events, allowing the genotypic data of both the father and son to be identical. Though in most cases a father-son pair will have the same Y-allelic data, random mutations like allele insertions and deletions can occur, which can interfere and result in incorrect conclusions in regards to paternity testing, forensic analysis, or genealogy. Though the exact mechanism of Y loci mutability is unknown, postulations of factors that can cause mutations have been studied, as well as attempts to determine mutation rate specific to each locus. A multi-generational pedigree consisting of 9 males was analyzed using two different methodologies: capillary electrophoresis and next-generation sequencing. The samples were amplified using either a ForenSeq™ Signature DNA Prep Kit (Verogen, San Diego, CA) or a YFiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific, Waltham, MA). Between the two methods, five Y-STR loci were identified as being discordant between a father-son pair. Next-generation sequencing identified an allele insertion at DYS385a/b, resulting in a potential tri-allelic locus, but was disproved after comparison with the capillary electrophoresis data of the sample. The capillary electrophoresis data identified four discordances between father-son pairs, one of which was an allele mutation with a gain of a repeat at DYS458. At DYS 389II, an allele insertion was identified, but was contradicted after comparison with the next-generation sequencing data. There was a potential null allele at DYS518 and either an OL variant allele or a 2 base pair deletion at DYS481. Following peak height ratio, stutter, and comparative analysis between the genotypic data of the two analysis methods, two of these discordances were proven to be errors, one was a definitive mutational event, and the other two could neither be confirmed nor denied due to differences in loci tested in each kit.

The United States (U.S.) government has been attempting to combat the growing opioid epidemic ravaging the nation. The opioid epidemic has had a significant impact on public health and forensic science laboratories. Moreover, this epidemic has moderate to fatal health consequences for expectant mothers with substance use disorder and their child who may develop Neonatal Abstinence Syndrome (NAS), otherwise known as Neonatal Opioid Withdrawal Syndrome (NOWS). The objective of this thesis is to emphasize that further research is needed for the identification and quantification of opioids in human breast milk. This topic has public health implications such as discussing the information gaps as it relates to a highly vulnerable group, women, and infants, affected by the opioid epidemic. Furthermore, there are implications in forensic science connected to postmortem toxicology and pathology when determining the cause of death and contributing factors in pediatric cases. This emphasis on the need for greater research will be accomplished by highlighting the opioid epidemic, its impact and further understanding of the addictive drug class known as opioids. The history of the crisis, effects on society as well as pharmaceutical knowledge of opioids will assist in development of plans to suppress growth and provide care for the afflicted. Furthermore, this thesis will attempt to demonstrate the need for further research involving opioids will be of significant value for public health and forensic science. As the forensic laboratories and various medical facilities are at the forefront of the opioid epidemic, there is a need for more robust, validated, inexpensive, and fast drug detection methodologies. Increasing rates of new designer drugs, addiction, and opioid-related deaths has caused a backlog in the forensic laboratories due to the great number of cases. While, the higher instances of maternal substance use disorder (SUD)/ opioid use disorder (OUD) with parallel increases in cases of NAS incidences are a few of the issues that need to be managed by public health leaders. Additionally, this thesis will examine current methodologies for drug quantification of opioids in human breast milk. The valid methodologies developed as well as the findings by the few available studies allowed for the current recommendations related to the acceptability of mothers in MAT programs, using methadone and buprenorphine during pregnancy and postpartum, being able to breastfeed their infant. By examining these studies and the findings, standardization criteria for the development of study designs for new methodologies relating to drug determination in human breast milk could be developed. The establishment of standardization criteria and acknowledging information gaps in current knowledge will be significant as these findings could influence policies, guidelines and procedures relating to maternal SUD/OUD, NAS/NOWS, and pediatric death determination as well as postmortem toxicology.

Traditionally, pre-contact Native Americans have served as a biological reference for identifying modern Asian individuals in aspects of the biological profile due to their distantly shared genetic history, although this assumption remains largely untested. This study explores the craniomorphic variability between Asian and Asian-derived groups to ascertain whether they can be differentiated using population-specific models. Cranial and mandibular nonmetric trait data were recorded on pre-contact Native Americans (n=150) and compared within a statistical framework to cranial trait data for modern Thai (n=150) and Japanese (n=150) individuals. Chi-square analyses indicate that the groups exhibit statistically significant differences in their trait expressions. Of the 35 traits analyzed, 31 differ significantly between the groups. Binary logistic regression equations for differentiating the Japanese, Thai, and Native Americans are presented, and cross-validated correct classification rates range 60.0-90.0%. Further, the inclusion of sex into the logistic regression equations failed to improve their accuracies. The results indicate that the Native American and Asian groups are not skeletally homogenous due to divergent population histories, and that numerous cranial and mandibular nonmetric traits are resolute enough to detect differences within and between Asian and Asian-derived groups. Thus, this study highlights the utility of nonmetric traits in identifying individuals beyond the traditional African (“Black”), (“White”), and Asian groupings.

Differential extraction has proven to be a challenging and time-consuming process, often requiring up to six hours of a forensic analyst’s concentration. With the ever-increasing backlog of sexual assault evidence kits, the forensic community is using new ways to diminish this backlog, including more streamlined evidence processing and sample analysis. The goals for processing sexual assault samples include efficient recovery of sperm deoxyribose nucleic acid (DNA), simplified sample processing, and the development of a profile eligible for forensic analysis. Cost and time can also be limiting factors. The Cotton Research Lab at Boston University has developed a novel method of differential extraction that combines separation of epithelial and sperm cell fractions, nuclease treatment to reduce female DNA carryover and a direct-cell lysis protocol. With the exception of a single centrifugation step, the entire protocol is conducted using a thermalcycler in the DNA extraction laboratory. Thus, the process is a Temperature Controlled Differential Extraction (TCDE), and has been effectively adapted for use with liquid, dried, and aged samples. The purpose of this research is to explore methods which further adapt the protocol for best use with forensic casework samples, namely vaginal swabs. Sexual assault evidence collection kits may contain a variety of items, and commonly include cotton swabs for the collection of fluids from intimate sources. To simulate casework-type samples, swabs were prepared with liquid epithelial cell preparations and various semen dilutions (ranging from 1:1 to 1:1000). Amendments were made to the TCDE protocol for best DNA recovery from a swab, and buffer changes were made to enhance compatibility with polymerase chain reaction (PCR)-amplification kits widely utilized in forensic labs. Finally, post-coital swabs from female donors were analyzed using the TCDE protocol with modifications for forensic casework samples. Preliminary testing of casework-type swabs with protocol modifications showed high yields of DNA and successful separation of epithelial and spermatozoa fractions. The epithelial fraction, when yielding a mixed profile, demonstrated a clear major female contributor, and the spermatozoa fractions showed little to no female carryover, often exhibiting single source male profiles. The TCDE protocol with modifications for casework-type samples requires approximately 2 hours and 30 minutes of an analyst’s time, from the moment the swab is removed from its evidence packaging to an extraction ready for DNA quant and short tandem repeat (STR) amplification. The method provides increased DNA recovery, can be used with various amplification kits, and generate probative profiles and is time efficient. This robust and promising new method that has the potential to be automated and to contribute to the effort to reduce the backlog in the analysis of sexual assault evidence kits.

The following is a comparison study of the Globalfiler™ PCR Amplification Kit analyzed on an ABI 3130 Genetic Analyzer Capillary Electrophoresis (CE) versus the ForenSeq™ DNA Signature Prep Kit analyzed on the MiSeq FGx™ System. The MiSeq FGx™ System measures results by Allele Read Count (ARC), while the CE measures results as Relative Fluorescent Units (RFU). Mixture samples were prepared in ratios of 1:1, 1:4, and 1:10 in replicates of four using a female major contributor and a male minor contributor, intended to represent some commonly seen mixture samples ratios in forensic cases [48]. Both systems performed equally well for the 1:1 mixture while the MiSeq FGx™ System had improved accuracy and precision for the 1:4 mixture compared to CE (4.033 + 1.506 ARC and 4.678 + 2.093 RFU, respectively). The MiSeq FGx™ System showed increased variation in the 1:10 mixture compared to CE (10.347 + 5.184 ARC and 9.311 + 3.363 RFU, respectively). Over the four replicates, the MiSeq FGx™ System had a total of 15 out of 528 possible alleles (2.84%) dropout compared to a total of 13 out of 384 possible alleles (3.39%) dropout on CE. The additional loci analyzed by the MiSeq FGx™ System results in a lower percentage of alleles lost due to dropout compared to CE. Isoalleles in sequence data may reveal the presence of minor contributor alleles that would otherwise be masked by the major contributor in length-based STR analysis. The presence of isoalleles are most helpful in mixture ratios greater than 1:1, where it is easier to assign alleles to a specific contributor. In certain cases, deconvolution of loci with shared alleles may not be improved by sequencing if intra-contributor isoalleles are present. Unless using known reference profiles, it is difficult to accurately assign alleles to contributors when intra-contributor isoalleles are present. Additionally, the sequencing data from the MiSeq FGx™ System provided information to aid the separation of stutter from true alleles. Previous studies report a significant increase in the amount of alleles present at some loci due to differences by nucleotide sequence, which may improve the discriminating power of those loci [31,52,53,54]. With all its potential, there is still much room for sequencing technology to improve before it becomes a standard analysis method in forensic laboratories.

Over time, criminals have become more aware of the different types of trace evidence that are capable of being identified by forensic analysis. As a result, the frequency of actions taken to prevent the transmission of evidence, specifically biological fluids and subsequent DNA evidence, with the usage of condoms in the commission of sexual assaults, has increased. With the increased use of condoms, comes the increased awareness and probative nature of forensic analysis of the potentially unique chemical profiles residues may leave behind. This includes the identification of lubricant type and of any additives that may be present, such as spermicides, flavoring or topical anesthetics. The two predominate condom lubricants are polydimethylsiloxane (PDMS) and polyethylene glycol (PEG): PEG, a water-soluble lubricant, is soluble in polar solvents, such as methanol, while PDMS, a silicone-based lubricant, is reported to be soluble in non-polar solvents, such as hexane. A total of thirty condoms representing eight brands, each of a different type, were evaluated by Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy (ATR-FTIR). It has been reported that PDMS is the more prevalent condom lubricant compared to PEG; this trend was reflected in this small subset of products. Of the thirty condom lubricants analyzed, twenty-five were PDMS (approximately 83%), six PEG (approximately 7%), six glycerol (approximately 7%) and one “other” (approximately 3%). A direct extraction method was developed to isolate the lubricant from the condoms. Following the direct extraction of the condom lubricants from ten condoms of different brands and types containing PDMS, the extraction capabilities of three solvents: hexane, methanol and methylene chloride, in the presence of blood and saliva separately, were evaluated. Two different biological fluid/lubricant sample types were created: liquid suspensions of lubricant, biological fluid and solvent; and contrived casework samples consisting of a mixture of lubricant and biological fluid dried onto a cotton swab. Hexane was capable of isolating only the PDMS lubricant in the presence of biological fluids. In an effort to extract and identify the water-soluble lubricant PEG and any additional additives, two Trojan condoms marketed as containing the spermicide nonoxynol 9 (N9) and one Durex condom marketed as containing the topical anesthetic benzocaine were obtained. Methanol was used as the direct extraction solvent as much of the published literature has determined that additives, such as spermicides and topical anesthetics, are often found in combination with PEG, which must be extracted in a polar solvent. Although capable of extracting the lubricant PEG, PDMS, and the N9 from the condoms directly, the presence of biological fluids prevented the successful isolation of any condom lubricants or additives with the use of methanol. This extraction study established the solubility of PDMS in both methanol and hexane as well as the limited solubility of PEG in methanol. To identify a solvent capable of extracting both lubricant types as well as the spermicide additive N9 in the presence of biological fluids, the extraction capabilities of methylene chloride were assessed. In the literature, methylene chloride is often used to eliminate a two-step, or two-solvent, extraction for condom lubricants. The isolation of PDMS and N9 had mixed results when using methylene chloride as a solvent. PDMS and N9 were successfully isolated and identified in one of the Trojan brand spermicidal condoms, but not the other, most likely due to a difference in concentration of N9 in the two condoms. Only PEG could be isolated in the Durex condom marketed as containing benzocaine using methylene chloride. In the blood and saliva/lubricant contrived casework samples extracted with methylene chloride, the PDMS and PEG in the respective condoms were isolated but N9 was not. An evaluation of solvent extraction efficiency was made by comparing the ability of each solvent to isolate condom lubricant and additives in the presence of biological fluids. Methylene chloride was found to be the most effective solvent when compared to hexane and methanol for this purpose.

Magister Scientiae Dentium - MSc(Dent) / Bite mark evidence has been admitted into US courts since the 1870s. It quickly gained popularity after the conviction of W.E. Marx in 1974 for manslaughter using primarily bite mark evidence. However, since the development of DNA typing and testing in forensic science, the emergence of wrongful convictions has placed the validity of bite mark evidence admissibility into severe dispute. This mini-thesis is a condensation of the past ten years’ worth of literature on the latest researches regarding bite mark evidence. The theory of the uniqueness of the human dentition is analysed. The accurate reproducibility of bite mark on skin with regard to distortion is discussed. Some bite mark court cases, including wrongful convictions are explored. Inconsistent expert opinions and the lack of standards amongst practitioners are also examined. The aim of this study is to summarize the validity of bite mark evidence in the courts of law.

Two deaths involving 3, 4-methylenedioxypyrovalerone (MDPV) are reported. MDPV is a synthetic cathinone stimulant found in "bath salts" with neurological and cardiovascular toxicity. Biological specimens were analyzed for MDPV by GC/MS and LC/MS. A White man was found dead with signs of nausea and vomiting after repeatedly abusing bath salts during a weekend binge. Femoral venous blood and urine had MDPV concentrations of 39 ng/mL and 760 ng/mL. The second fatality was a White man with a history of drug and bath salt abuse found dead at a scene in total disarray after exhibiting fits of anger and psychotic behavior. Femoral venous blood and urine had MDPV concentrations of 130 ng/mL and 3800 ng/mL. The blood and urine MDPV concentrations are within the reported recreational concentration ranges (blood 24-241 ng/mL and urine 34-3900 ng/mL). Both decedents' deaths were attributed to relevant natural causes in a setting of MDPV abuse.

Two deaths involving 3, 4-methylenedioxypyrovalerone (MDPV) are reported. MDPV is a synthetic cathinone stimulant found in "bath salts" with neurological and cardiovascular toxicity. Biological specimens were analyzed for MDPV by GC/MS and LC/MS. A White man was found dead with signs of nausea and vomiting after repeatedly abusing bath salts during a weekend binge. Femoral venous blood and urine had MDPV concentrations of 39 ng/mL and 760 ng/mL. The second fatality was a White man with a history of drug and bath salt abuse found dead at a scene in total disarray after exhibiting fits of anger and psychotic behavior. Femoral venous blood and urine had MDPV concentrations of 130 ng/mL and 3800 ng/mL. The blood and urine MDPV concentrations are within the reported recreational concentration ranges (blood 24-241 ng/mL and urine 34-3900 ng/mL). Both decedents' deaths were attributed to relevant natural causes in a setting of MDPV abuse.

The present study involves a multivariate assessment of the success of evidence recovery by searchers from various backgrounds and skill levels. Volunteers representing four experience levels (civilian volunteers, first year forensic anthropology graduate students, second year forensic anthropology graduate students, and first responders) conducted line searches of mock crime scenes, flagging items of forensic significance with pin flags. The groups were then briefly trained in human skeletal remains recovery, and implemented this training through a second set of mock scene searches. Recovery rates were compared across pre- and post-training trials and across searcher groups in order to determine the influence of searcher training and experience on search success. The results of this study reveal not only the percentage of evidence that was recovered by search teams, but exhibits the degree to which experience and training played a role in evidence recovery.