Ultrafast STR Separations on Short‐Channel Microfluidic Systems for Forensic Screening and Genotyping

There are situations in which it is important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. DNA typing methods provide the best biometric information yielding identity, kinship, and geographical origin, but they are not portable and rapid. This study details the development of a portable short‐channel microfluidic device based on a modified Agilent 2100 bioanalyzer for applications in forensic genomics. The system utilizes a denaturing polymer matrix with dual‐channel laser‐induced fluorescence and is capable of producing a genotype in 80 sec. The device was tested for precision and resolution using an allelic ladder created from 6 short tandem repeat (STR) loci and a sex marker (amelogenin). The results demonstrated a precision of 0.09–0.21 bp over the entire size range and resolution values from 2.5 to 4.1 bp. Overall, the results demonstrate the chip provides a portable, rapid, and precise method for screening amplified short tandem repeats and human identification screening.     

A mixed DNA profile controversy revisited

Semaan et al. (J Forensic Res, 2020, 11, 453) discuss a mock case “where eight different individuals [P₁ through P₈] could not be excluded in a mixed DNA analysis. Even though … expert DNA mixture analysis software was used.” Two of these are the true donors. The LRs reported are incorrect due to the incorrect entry of propositions into LRmix Studio. This forced the software to account for most of the alleles as drop-in, resulting in LRs 60–70 orders of magnitude larger than expected. P₁, P₂, P₄, P₅, and P₈ can be manually excluded using peak heights. This has relevance when using LRmix which does not use peak heights. We extend the work using the same two reference genotypes who were the true contributors as Semaan et al. (J Forensic Res, 2020, 11, 453). We simulate three two-donor mixtures with peak heights using these two genotypes and analyze using STRmix?. For the simulated 1:1 mixture, one of the non-donors’ LRs supported him being a contributor when no conditioning was used. When considered in combination with any other potential donors (i.e., with conditioning), this non-donor was correctly eliminated. For the 3:1 mixture, all results correctly supported that the non-donors were not contributors. The low-template 4:1 mixture LRs with no conditioning showed support for all eight profiles as donors. However, the results from pair-wise conditioning showed that only the two ground truth donors had LRs supporting that they were contributors to the mixture. We recommend the use of peak heights and conditioning profiles, as this allows better sensitivity and specificity even when the persons share many alleles.     

STR Genotyping from a Dry‐Cleaned Skirt in a Sexual Assault Case

In this sexual assault case, the standard preliminary semen examinations could not confirm physically or biochemically whether the accused's semen had stained the victim's skirt because the skirt had been dry‐cleaned for stain removal and had been worn for more than a year after the assault. Fortunately, however, a photograph taken just after the assault was found in the court records that showed white stains on the checkered skirt. The locations of the stains were estimated based on the checkered pattern of the fabric, and microscopic examination using Baecchi's staining revealed the presence of spermatozoa. Further analysis indicated the male DNA profile generated from the sperm cells was consistent with the suspect's DNA using three multiplex STR typing systems for a total of 21 autosomal and 17 Y chromosomal short tandem repeats (STRs). Ultimately, the result of the DNA profile played a very useful role as additional evidence.     

Cold Case Homicides: DNA Testing of Retained Autopsy Sexual Assault Smears

Archival medical examiner specimens may contain perpetrator DNA evidence useful in unsolved (“cold case”) homicides. The Office of the Chief Medical Examiner (OCME) histology slide archives were searched for sexual assault smears for all 376 female homicides from 1990 to 1999. Of these, the OCME had sexual assault smears on 84 of which 13 slides had sperm. Of these 13, six were still unsolved. DNA profiles were obtained on all six (5 from smears and one from swabs). Combined DNA Index System ( submission resulted in two matches (“hits”) for new suspects. In addition, three suspects were eliminated in two cases. Our review of archival sexual assault smears resulted in DNA profiles that were able to assist in the investigation of four cold case homicide investigations. It may be worthwhile for medical examiner offices to search their archival histology slides for sexual assault smears on previously unsolved cases particularly those prior to the mid‐1990s when DNA testing was less widely available.     

Collecting and Analyzing DNA Evidence from Fingernails: A Comparative Study,,

Forensic practitioners and crime laboratories regularly collect and analyze fingernail evidence; however, the best techniques for processing such evidence have not been established. In this study, numerous aspects of fingernail evidence processing—collection of exogenous cells, transportation, purification of DNA, and STR analysis—were analyzed using fingernails harboring applied blood or epithelial cells from scratchings. Autosomal STR mixtures resulted when fingernails were soaked or swabbed, while scrapings rarely generated mixtures but exhibited allelic dropout. Y‐STRs yielded single source profiles, with scrapings again showing dropout. A silica‐based kit extraction recovered significantly more exogenous DNA than did organic extraction, neither of which was affected by nail polish. Swabbing nails in succession resulted in some cross‐contamination from exogenous material, while transporting nails together did not, although there was loss of exogenous cells. Optimized nail processing produced complete Y‐STR profiles of male volunteers from female fingernails following scratchings.     

The Use of Forensic Tests to Distinguish Blowfly Artifacts from Human Blood,Semen, and Saliva

This study investigated whether routinely used forensic tests can distinguish 3‐day‐old or 2‐week‐old fly artifacts, produced after feeding on human blood, semen, or saliva, from the biological fluid. Hemastix^®, Hemident^?, and Hemascein^? were unable to distinguish blood from artifacts. Hemastix^® returned false positives from negative controls. ABAcard^® Hematrace^® and Hexagon OBTI could distinguish blood from 3‐day‐old artifacts, but not 2‐week‐old artifacts. Phadebas^® and SALIgAE^® were unable to distinguish saliva from artifacts. RSID^?‐Saliva was able to distinguish saliva from 3‐day‐old artifacts, but not 2‐week‐old artifacts. Semen tests Seminal Acid Phosphatase, RSID^?‐Semen, and ABAcard^® p30 were all able to distinguish semen from 3‐day‐old artifacts, but not 2‐week‐old artifacts. The tests investigated cannot be relied upon to distinguish artifacts from biological fluids. However, if an artifact is identified by its morphology, a positive result may indicate which biological fluid the fly consumed, and this knowledge may prove useful for investigators searching for DNA.     

Identification of Missing Norwegian World War II Soldiers,in Karelia Russia

This article presents the multidisciplinary effort in trying to identify the skeletal remains of 100 Norwegian soldiers serving in the German army, killed in Karelia Russia in 1944, from the recovery of the remains through the final identification using DNA. Of the 150 bone samples sent for DNA testing, 93 DNA profiles were obtained relating to 57 unique individuals. The relatives could not be directly contacted as the soldiers were considered as traitors to Norway; therefore, only 45 reference samples, relating to 42 cases of the missing, were donated. DNA matches for 14 soldiers and 12 additional body part re‐associations for these individuals were found. Another 24 bone samples were re‐associated with 16 individuals, but no familial match was found. More than six decades after the end of WWII, DNA analysis can significantly contribute to the identification of the remains.     

Error and its Meaning in Forensic Science

The discussion of “error” has gained momentum in forensic science in the wake of the Daubert guidelines and has intensified with the National Academy of Sciences' Report. Error has many different meanings, and too often, forensic practitioners themselves as well as the courts misunderstand scientific error and statistical error rates, often confusing them with practitioner error (or mistakes). Here, we present an overview of these concepts as they pertain to forensic science applications, discussing the difference between practitioner error (including mistakes), instrument error, statistical error, and method error. We urge forensic practitioners to ensure that potential sources of error and method limitations are understood and clearly communicated and advocate that the legal community be informed regarding the differences between interobserver errors, uncertainty, variation, and mistakes.     

A Novel Method for Identifying Shahtoosh

Shahtoosh, the down hair of the Tibetan antelope (Pantholops hodgsonii), is the noblest and most expensive wool in the world. The population of the animal has declined dramatically due to commercial poaching for the fiber. Traditional inspection for detection of shahtoosh has been performed by microscopic analysis. We developed a TaqMan real‐time PCR‐based DNA analysis method for identifying shahtoosh fibers. A set of probe and primers for the mitochondrial 12S ribosomal RNA gene that binds specifically to Tibetan antelope DNA was designed. A signal was detected with sensitivity to the 1:10,000 dilution of shahtoosh DNA. A fiber mixture of 1% of shahtoosh mixed with cashmere and even a single fiber can be detected with this method. The method is faster, more cost‐effective and more sensitive than other traditional sequencing methods and can be directly applied to identify shahtoosh and its processed products, which will be of value in illegal trade investigations.     

DNA Profiles from Fingerprint Lifts—Enhancing the Evidential Value of Fingermarks Through Successful DNA Typing

This study evaluated the compatibility of the most common enhancement methods and lifting techniques with DNA profiling. Emphasis is placed on modern lifting techniques (i.e., gelatin lifters and Isomark?) and historical fingerprint lifts for which limited research has been previously conducted. A total of 180 fingerprints were deposited on a glass surface, enhanced, lifted, and processed for DNA typing. DNA could be extracted and profiled for all the powders and lifts tested and from both groomed fingerprints and natural prints with no significant difference in the percentage of profile recovered. DNA profiles could also be obtained from historical fingerprint lifts (79.2% of 72 lifts) with one or more alleles detected. These results demonstrate the compatibility between different powder/lift combinations and DNA profiling therefore augmenting the evidential value of fingerprints in forensic casework.     

Radiation safety for the NOMAD portable X-ray system in a temporary morgue setting

The purpose of this study was to determine the radiation levels resulting from leakage and scatter encountered by the forensic dental personnel using the Nomad at St. Gabriel, LA, following Hurricane Katrina. Using a Keithley Radiation Survey Meter and Lucite head phantom, radiation levels were measured at various distances and angles from the Nomad corresponding to the positions occupied by the dental personnel at St. Gabriel. The measurements were used to approximate the maximum total radiation dose from the Nomad to each team member for a 2- and a 4-week deployment. The results show that the maximum scatter radiation dose to any team member was 4.4 microR per X-ray or 0.253 millisieverts (mSv) for a 2-week deployment and 0.506 mSv for a 4-week deployment. Therefore, the leakage and scatter radiation dose from the Nomad was insignificant compared with established radiation safety guidelines of 50 mSv per year for all team members.     

The Food Preferences of the Blow Fly Lucilia cuprina Offered Human Blood,Semen and Saliva,and Various Nonhuman Foods Sources

As human DNA profiles can be obtained from blow fly artifacts, this study aimed to establish the feeding preferences of Lucilia cuprina (Wiedemann) blow flies when offered human biological fluids and nonhuman food sources. One‐day‐old and 3‐day‐old blow flies of both sexes were simultaneously offered human blood, semen and saliva, pet food, canned tuna and honey, and the number and length of visits documented over 6 h. One‐day‐old flies visited pet food and honey most often, but stayed longest on honey and semen. Three‐day‐old flies visited semen and pet food most often, and stayed longest on these food sources. Blood and saliva were the least preferred options for all flies. Overall, flies preferred dry blood and semen to the wet forms. These findings demonstrate that even when other food sources are available, flies at a crime scene may feed on human biological fluids if present, potentially transferring human DNA.     

DXAGE: A New Method for Age at Death Estimation Based on Femoral Bone Mineral Density and Artificial Neural Networks

Age at death estimation in adult skeletons is hampered, among others, by the unremarkable correlation of bone estimators with chronological age, implementation of inappropriate statistical techniques, observer error, and skeletal incompleteness or destruction. Therefore, it is beneficial to consider alternative methods to assess age at death in adult skeletons. The decrease in bone mineral density with age was explored to generate a method to assess age at death in human remains. A connectionist computational approach, artificial neural networks, was employed to model femur densitometry data gathered in 100 female individuals from the Coimbra Identified Skeletal Collection. Bone mineral density declines consistently with age and the method performs appropriately, with mean absolute differences between known and predicted age ranging from 9.19 to 13.49 years. The proposed method—DXAGE—was implemented online to streamline age estimation. This preliminary study highlights the value of densitometry to assess age at death in human remains.     

The Morphology of Fecal and Regurgitation Artifacts Deposited by the Blow Fly Lucilia cuprina Fed a Diet of Human Blood

Fly feces and regurgitation deposits may be mistaken for bloodstain patterns at a crime scene, potentially compromising event reconstruction and/or misdirecting police resources. In some instances, these artifacts contain sufficient human biological material to generate a full DNA profile, sometimes 2 years after deposition. Clearly, it is important that investigators can make the distinction between artifacts and bloodstains. This study examined 6645 artifacts deposited on a smooth, nonporous surface after Lucilia cuprina were fed human blood. Artifacts were also compared with bloodstains on a variety of other surfaces. Both similarities and differences were found between artifacts and bloodstains, highlighting the need for an identification system to assist personnel with little training in bloodstain pattern analysis. The morphology of the artifacts has been described so that these deposits may be more clearly distinguished from bloodstains, targeted by crime scene personnel as potential sources of human DNA, and/or identified as potential evidence contaminants. Flowcharts have been devised to facilitate the analysis.     

The Effect of Flunitrazepam (Rohypnol®) on the Development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) and its Implications for Forensic Entomology

This study investigated the potential effects of flunitrazepam (known as “date rape drug”) on the developmental cycle of Chrysomya megacephala, an important forensic species, and their possible implications for the calculation of the PMI. A 1050 C. megacephala eggs were divided into five groups with seven replications each. The eggs were placed on artificial diet prepared with four drug concentrations of flunitrazepam (4, 8, 16, and 32 ng/g), besides the control group (prepared with water). Were evaluated the potential effects on development time, weight gain, and mortality during the cycles. The drug had no significant effect on development time or mortality although it did affect the weight of the pupae and adults (Kruskal–Wallis, p < 0.05). The result can be deduced that the determination of the postmortem interval is not affected.     

Applicability of the ParaDNA® Screening System to Seminal Samples

Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA^® Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework.     

High‐Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high‐resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual‐dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics.     

An Optimized Centrifugal Method for Separation of Semen from Superabsorbent Polymers for Forensic Analysis

Connection of a perpetrator to a sexual assault is best performed through the confirmed presence of semen, thereby proving sexual contact. Evidentiary items can include sanitary napkins or diapers containing superabsorbent polymers (SAPs), complicating spermatozoa visualization and DNA analysis. In this report, we evaluated the impact of SAPS on the current forensic DNA workflow, developing an efficient centrifugal protocol for separating spermatozoa from SAP material. The optimized filtration method was compared to common practices of excising the top layer only, resulting in significantly higher sperm yields when a core sample of the substrate was taken. Direct isolation of the SAP‐containing materials without filtering resulted in 20% sample failure; additionally, SAP material was observed in the final eluted DNA samples, causing physical interference. Thus, use of the described centrifugal‐filtering method is a simple preliminary step that improves spermatozoa visualization and enables more consistent DNA yields, while also avoiding SAP interference.     

Species Identification of Cannabis sativa Using Real‐Time Quantitative PCR (qPCR),

Most narcotics‐related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3′ exon‐trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real‐time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa.