Forensic biology encompasses the examination of evidentiary items from crime scenes for biological fluids, often identifying the specific biological fluid present and developing a DNA profile that can be used to link a suspect to a crime. Blood identification consists of visual examination, presumptive tests based on the catalytic activity of hemoglobin, and confirmatory tests based on antigen-antibody interactions. Issues encountered in blood identification include the occurrence of false positive and false negative results. Many causes of these results are well-known but more recently three substances resulting in false negatives with catalytic color tests, chemiluminescent reagents, and immunoassays have been explored. Quebracho extract (a common leather tannin), sodium percarbonate (the main component of detergents containing active oxygen) and vitamin C-containing beverages were all found to produce false negative results at varying degrees with each of the tests mentioned. Increased knowledge of potential negative interfering agents by forensic investigators can help ensure that probative evidence is properly collected and thoroughly analyzed from a crime scene.

Blood is considered one of the most important types of forensic evidence found at a crime scene. The use of surface-enhanced Raman spectroscopy (SERS) provides a potentially non-destructive and highly sensitive technique for the confirmation of blood and this method can be applied using a portable Raman device with quick sample preparation and processing. Crime scenes are inherently complex and the impact of SERS analysis provides easy use and practical application for in-field sample analysis. SERS is one of the few confirmatory techniques employed for the identification of blood at a crime scene or in the forensic laboratory. This method is able to distinguish between blood and other body fluids by collecting a SERS spectrum from a sample placed on a surface that has been embedded with gold nanoparticles (AuNPs). The AuNPs create an electric field surface enhancement that produces an intense molecular vibrational signal, leading to a SERS enhancement. The SERS enhancement allowed for sensitive blood detection at dilutions greater than 1:10,000. A stain transfer method to the SERS substrate was optimized by extracting dried bloodstains with water, saline, and various acid solutions. Fifty percent aqueous acetic acid solutions was found to be the most efficient in retaining the blood components and releasing the hemoglobin component of blood for detection. The SERS spectrum of blood is a robust signature of hemoglobin that does not significantly change between donors nor over time. Characteristic peaks for the identification of blood are 754, 1513, and 1543 wavenumbers (cm-1), attributed to a pyrrole ring breathing mode (15) and two Cβ-Cβ stretches (11, 38), respectively. These key SERS peaks, high sensitivity, and signal enhancement are favorable when compared to normal Raman spectroscopy. A quick and easy-to-use procedure for on-site sample analysis for the detection of blood on different substrates was developed and applied on a portable Raman device. Various nonporous and porous substrates including glass, ceramic tile, cotton, denim, fleece, nylon, acetate, wool, polyester, wood, and coated wood yielded strong results for identification of bloodstains. In addition, different commercial and in-house SERS substrates were tested to determine effectiveness for the detection and identification of blood. SERS identification of blood for forensic work is a potentially non-destructive and portable tool that can be applied for quick and easy examination of evidence at a crime scene. The high sensitivity and selectivity of SERS provides a robust spectroscopic signature that aids in the confirmation of blood, even when it is not visible to the naked eye. It is a more favorable method when compared to current presumptive and confirmatory tests for blood and can be applied to stains on different SERS substrates and a variety sample surfaces for universal testing.

Blood is considered one of the most widely tested biological matrices. The first step in blood identification involves visual examination followed by presumptive testing. Once a positive presumptive result is observed, confirmatory tests are performed to determine that a stain is human blood, thus reducing the time and resources spent on forensically irrelevant samples. When interfering agents are present, this general workflow is hindered as presumptive tests can render false-negative results. General awareness of these interfering agents can help analysts to recognize forensically relevant evidence that may have otherwise been deemed immaterial. The main objective of this study was to understand various interfering agents and their effects on presumptive blood tests such as Kastle Meyer (KM) and Orthotolidine (O-tol) reagents and confirmatory tests such as HemaTrace® and Rapid Stain Identification (RSID™) Blood. Additional experiments explored the effects on downstream DNA analysis. In the first part of the study, bloodstains in varying concentrations were exposed to ten chemical interferents over a period of time to understand how blood dilution, age of the stain, and the chemical nature of the interferent affect presumptive blood test results. Antioxidants, active oxygen, and tannins are known to interrupt the mechanism of presumptive tests. Thus, ten interfering agents (ascorbic acid, chlorogenic acid, catechin, sodium percarbonate, hydrogen peroxide, oxalic acid, proanthocyanidins, quebracho extract, chestnut extract, and theaflavin) were selected based on these characteristics. Six blood dilutions (neat, 1:10, 1:50, 1:100, 1:500, and 1:1000) were exposed to the interferents, and presumptive tests for blood were conducted on six days (day 1, 8, 22, 43, 71, and 106). The second part of the study examined bloodstains deposited on real-world samples (wines, citrus fruit juices, teas, coffee, cleaning agents, and leather products) containing chemical interferents. In addition, confirmatory testing for human blood was conducted with HemaTrace® and RSID™-Blood on day 106 using the 1:500 dilution. Finally, DNA analysis of 1:10 dilution stains was performed on day 150 to study whether downstream DNA analysis was compromised due to the presence of the interferents. The results showed that as blood concentration reduced, more false-negative results were observed when chemical interferents were present. Further, chemical interferents produced frequent atypical color changes in tests with KM and O-tol reagents, while only some atypical color changes were observed with the household products tested. Immunochromatographic assay results indicated both HemaTrace® and RSID™-Blood could detect the presence of blood when interfering agents are present, although the positive result bands with RSID™-Blood were faint and sometimes difficult to visualize. Poor DNA results from the untreated blood sample limited any interpretation of the DNA results obtained from bloodstains deposited on household products. Overall, the data indicates that valuable blood evidence may be overlooked due to faint or false-negative results when these interferents are present. Future studies should focus on how these interferents may affect downstream DNA analysis.

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<p>  </p> <p>Blow flies are often utilized in the field of forensic science due to their ability to aid in the estimation of time since death. Currently, estimations of postmortem interval require assumptions to be made and are prone to a margin of error, prompting research that may contribute to more accurate postmortem interval estimations and help to fill in the gaps of unknown information. Blow flies are necrophagous, feeding on feces and carrion, and therefore, are constantly sampling the environment. This behavior can be exploited in order to monitor the biodiversity in an environment. Through analysis of DNA isolated from the guts of blow flies, information can be obtained regarding what animals have died in an environment, what animals are still living in that environment, and the abundance and diversity of the animals present in a specific environment. Using fly-derived ingested DNA is a viable method for vertebrate resource identification and biodiversity monitoring. Over the course of a two-summer sampling period, in and around two national parks, a total of 162 blow fly (<em>Phormia regina</em>) samples returned a positive vertebrate DNA identification, with 33 species identified from five animal orders. Of the total number of flies collected and analyzed, 23.58% returned a positive vertebrate species identification. The method detected both abundant and common species based on National Park surveys, as well as some uncommon or unknown to the park species. In the SE region, 9 individuals belonging to the Rodentia order, 12 individuals belonging to the Artiodactyla order, 21 individuals belonging to the Carnivora order, 1 individual belonging to the Cingulata order, and 3 individuals belonging to the Lagomorph order were detected. In the SE region, 63% of the individuals detected belonged to the common category, 14% of the individuals detected belonged to the uncommon category, and 23% of the individuals detected belonged to the not in park/unknown category. In the NW region, 42 individuals belonging to the Rodentia order, 46 individuals belonging to the Artiodactyla order, and 28 individuals belonging to the Carnivora order were detected. In the NW region, 52% of the individuals detected belonged to the abundant category, 36% of the individuals detected belonged to the common category, and 12% of the individuals detected belonged to the uncommon category. The relative biodiversity of the sampled environment can be inferred. In the SE region, the Shannon Biodiversity Index was calculated to be 2.28 with an evenness of 0.844, while in the NW region, the Shannon Biodiversity Index was calculated to be 2.79 with an evenness of 0.855. Unsurprisingly, there was greater biodiversity in the Northwest Park samples than in the Southeast Park samples. Additionally, the ideal weather conditions for blow fly collection were determined be at a temperature of between 60- and 80-degrees Fahrenheit, a relative humidity between 50% and 60%, no precipitation, and a wind speed between 2 and 8 miles per hour. This information has further implications in the field of forensic science, specifically dealing with wildlife forensics, pathogen distributions, and can help to improve accuracy in regards to postmortem interval (PMI) estimations. </p> <p>  </p>

Thousands of sexual assault cases in the United States are backlogged. This has been a growing issue for years that has increased the difficulty of solving these cases and providing closure to the victims. The analysis process for each case includes the identification of body fluids, presumptive testing, confirmatory testing, and DNA extraction. The only confirmatory method for semen identification is a microscopic visualization of sperm cells. The time spent on microscopic analysis varies depending on the complexity of the samples and the skills of the analyst. While the identification of sperm cells is informative, it can be very time-consuming and labor intensive. Some forensic laboratories choose to skip this step and submit samples directly for DNA analysis. Conducting DNA analysis on unscreened samples can increase the cost of testing when negative samples are analyzed as well as the time it takes to process each case. Automated microscopy has been available for decades and more recently has been paired with artificial intelligence to detect sperm cells on microscope slides. In this research, the MetaSystems automated microscope was used to analyze slides that mimic forensic sexual assault samples. Slides were also examined using traditional microscopy. The automated system quickly provided an accurate quantification of the number of sperm cells present in a sample, which can inform downstream DNA testing. The software was successful in identifying sperm cells treated with Christmas tree and hematoxylin and eosin stains, even among epithelial cells and various contaminants. Results demonstrated that an artificial intelligence-driven forensic sperm cell detection microscope can significantly reduce the time it takes to locate and identify sperm cells and estimate sperm cell quantity compared to a lengthier and more tedious manual search. Drawbacks to the system include the relatively high cost and reduced ability to accurately detect sperm cells amid contaminants that are of similar morphology.

<p dir="ltr"><i>Phormia regina</i> Meigen is a forensically relevant species of blow fly, common in North America and used to estimate the minimum postmortem interval in forensic casework. It is also possible to use blow flies to survey the environment for biotic and abiotic information drawn from both larval and adult stages. There are both forensic and environmental uses for genetic analysis of blow flies. Blow fly kinship is especially useful for detecting postmortem movement of a corpse or to assess abundance of carrion in a given location. To test genetic relationships between individuals, discriminatory loci such as microsatellites, or polymorphic tandemly repeated sequences of DNA are necessary. Here, we characterize novel microsatellites generated from the genome of <i>P. regina</i>. Thirty-four candidate polymorphic loci with conserved flanking regions, have been isolated. To date, seven are heterozygous and polymorphic testing in two lab populations and one wild population. The simple sequence repeats characterized here complement existing loci (N = 6) for greater discrimination for testing relationships between individual flies.</p>

<p dir="ltr">Blow flies are a family of carrion insects that are among the first to arrive in the decomposition process. Blow flies are known to ingest carrion, feces, water, and occasionally nectar to meet nutritional requirements. These behaviors make blow flies a unique organism potentially containing genetic material from a variety of sources within one environment. Their global distribution and ease of capture makes them a strong candidate for resource monitoring and identification. While previous studies have evaluated the suitability of blow flies for vertebrate biodiversity estimates, no work has been done looking at their ability to ingest and store genetic material from plants and microbes present in water. It is also not known how long these DNA signals persist in the gut. Through DNA analysis of the blow fly gut, researchers can identify vertebrates that have recently died in an environment, what plant species are present, and what water source the insect utilized. Through lab colony (Phormia regina) feeding experiments, it was determined that at 25 ˚C and 50 % relative humidity, vertebrate and plant DNA persist in the gut for over 120 hours post-ingestion. Wild sample analysis of flies collected from Yellowstone National Park was performed to identify plant species ingested by P. regina in the wild. Following Sanger sequencing, top hits on BLASTn included Brassicales, Juglans cathyensis, and uncultured Candida. This is the first application of environmental DNA analysis techniques to insects for the purpose of plant identification. This work also attempts to characterize microbial profiles of the gut of P. regina for the purpose of water resource identification. Over a two-month collection period, samples were collected from different water resources across Indianapolis. Flies were exposed to these samples in a controlled feeding experiment, followed by sampling at 0- and 72-hours post-exposure. Gut samples were sequenced using Illumina and Operational Taxonomic Unit clustering grouped reads by sequence similarity for identification. Bacteria classes identified included Gammaproteobacteria, Bacteroidia, Flavobacteria, Alphaproteobacteria, Bacilli, Clostridia, Actinobacteria, Betaproteobacteria, and Fusobacteria. Many bacteria classes were common across water samples, although the abundance of each class changed between samples and across time. These unique microbial profiles can be used to identify water resources for potential contamination and chemical dumping. Further work is necessary to generate microbial profiles from the original water sources themselves and for generation of alpha and beta diversities. Overall, this work spans multiple fields. Species identification is important for biodiversity monitoring and environmental surveys. Utilizing blow fly derived DNA allows for detection of living and deceased vertebrates in an environment, plant life, and water quality within one sample. This work also has implications in forensic science, specifically wildlife forensics and chemical detection of clandestine laboratories and chemical weapon compounds.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Forensic DNA profiling is widely used as an identification tool for associating an individual with evidence of a crime. Analysis of a DNA sample involves observation of data in the form of an electropherogram, and subsequently annotating a DNA “profile” from an individual or from the evidence. The profile obtained from the evidence can be compared to reference profiles deposited in a national DNA database, which may include the potential contributor. Following a match, a random match probability is calculated to determine how common that genotype is in the population. This is the probability of obtaining that same DNA profile by sampling from a pool of unrelated individuals. Each state has adopted various laws requiring suspects and/or offenders to submit a DNA sample for the national database (such as California’s law that all who are arrested must provide a DNA sample). These profiles can then be associated with past unsolved crimes, and remain in the database to be searched in the event of future crimes. In the case of database samples, a physical sample of the offender’s DNA must be kept on file in the laboratory indefinitely so that in the event of a database hit, the sample is able to be retested. Current methods are to collect a buccal swab or blood sample, and store the DNA extracts under strict preservation conditions, i.e. cold storage, typically -20° C. With continually increasing number of samples submitted, a burden is placed on crime labs to store these DNA extracts. A solution was required to help control the costs of properly storing the samples. FTA™ paper was created to fulfill the need for inexpensive, low maintenance, long term storage of biological samples, which makes it ideal for use with convicted offender DNA samples. FTA™ paper is a commercially produced, chemically treated paper that allows DNA to be stored at room temperature for years with no costly storage facilities or conditions. Once a sample is required for DNA testing, a small disc is removed and is to be used directly in a PCR reaction. A high quality profile is important for comparing suspect profiles to unknown or database profiles. A single difference between a suspect and evidentiary sample can lead to exclusion. Unfortunately, the DNA profile results yielded from the direct addition have been unfavorable. Thus, most crime laboratories will extract the DNA from the disc, leading to additional time and cost to analyze a reference sample. Many of the profiles from the direct addition of an FTA™ disc result in poor quality profiles, likely due to an increase in PCR inhibitors and high concentrations of DNA. Currently, standardized protocols regarding the recommended locations for removal of a sample disc from a bloodspot on an FTA™ card does not exist. This study aims to validate the optimal location by comparing DNA profiles obtained from discs removed from the center, halfway, and edge locations of a bloodspot from 50 anonymous donors. Optimal punch location was first scored on the number of failed, partial or discordant profiles. Then, profile quality was determined based on peak characteristics of the resulting DNA profiles. The results for all three disc locations were 5.3% failed amplifications, 4.2% partial amplifications, and one case of a discordant profile. Profile quality for the majority of the samples showed a high incidence of stutter and the absence of non-template adenylation. Of the three disc locations, the edge of the blood stain was ideal, due to a presumably lower concentration of DNA and likely more dilute amount of the PCR inhibitor heme. Therefore, based on the results of this study, there is a greater probability of success using a sample from the edge of a blood stain spotted in FTA™ paper than any other location of the FTA™ card.