Preparation of Artificial Blood from the Extract of Legume Root Nodules,and the Creation of Artificial Latent Fingermarks in Blood Using Artificial Blood,

Distribution of homogeneous fingermarks in blood is essential for conducting proficiency tests in forensic science. Hence, the artificial blood was prepared using the root nodule extract of Glycine max plants. The reactivity of the artificial blood with widely used human blood detection reagents was tested. Artificial latent fingermarks in blood were printed using an inkjet cartridge case filled with artificial blood solution. The artificial latent fingermarks in blood were developed with amino acid‐sensitive reagents and could obtain development as prominent as the image of the master fingermark saved on the computer. Therefore, it has been confirmed that the extract of legume root nodules can be used as artificial blood, and the artificial blood can be used for the preparation of artificial latent fingermarks or footmarks in blood.     

Simultaneous Identification of Four “Legal High” Plant Species in a Multiplex PCR High‐Resolution Melt Assay,

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus^®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.     

Evaluating the Efficiency of Primer Extension Capture as a Method to Enrich DNA Extractions,

In this study, we sought to document the efficiency of primer extension capture (PEC) as a method to enrich DNA eluates of targeted DNA molecules and remove nontarget molecules from pools containing both. Efficiency of the method was estimated by comparing number of “copies in” to “copies out” by quantitative polymerase chain reaction. PEC retention of DNA targets ranging 109‐288 base pairs (bps) in length was 15.88–2.14% (i.e., loss of 84.12–97.86% of target molecules). Experimental modifications of the PEC method resulted in no significant improvements. However, the benefit of PEC was revealed in its ability to remove most nontarget DNA molecules (99.99%). We also discovered that many (56.69%) of the target molecules are “lost” prior to their immobilization on the streptavidin‐coated beads. These estimates of methodological efficiency are directly comparable to previous ones observed following “fishing” for DNA, an alternative method for DNA enrichment.     

Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.     

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.     

“YFlag” – A Single‐base Extension Primer Based Method for Gender Determination

Assigning the gender of a DNA contributor in forensic analysis is typically achieved using the amelogenin test. Occasionally, this test produces false‐positive results due to deletions occurring on the Y chromosome. Here, a four‐marker “YFlag” method is presented to infer gender using single‐base extension primers to flag the presence (or absence) of Y‐chromosome DNA within a sample to supplement forensic STR profiling. This method offers built‐in redundancy, with a single marker being sufficient to detect the presence of male DNA. In a study using 30 male and 30 female individuals, detection of male DNA was achieved with c. 0.03 ng of male DNA. All four markers were present in male/female mixture samples despite the presence of excessive female DNA. In summary, the YFlag system offers a method that is reproducible, specific, and sensitive, making it suitable for forensic use to detect male DNA.     

Improving Luminol Blood Detection in Forensics

The aim of this study was to develop chemical improvements to the original Weber protocol, in order to increase the intensity and time length of light emission and to eliminate false‐positive reactions. The intensity and duration of light were measured on serial blood dilutions using a plate reader chemiluminometer. Blood stains of various concentrations were impregnated in pure cellulose, dried, and luminol solution was added with/without the potential enhancers. An in silico study was also conducted, aiming to demonstrate the enhancing mechanism of hemoglobin denaturation using 8 M urea. The luminol blood detection test revealed important improvements after urea pretreatment or in the presence of monochloro‐triazinyl‐β‐cyclodextrin. This approach also eliminated the false‐positive reaction from sodium hypochlorite. These improvements could provide a higher sensitivity under particular circumstances such as old or washed blood stains, leading to a better localization for further DNA typing and higher quality photographic analysis.     

National Academy of Sciences “Standardization”: On What Terms?

The frequently cited 2009 National Academy of Sciences Report entitled “Strengthening Forensic Science in the United States: A Path Forward” has become a focal point of forensic science practitioners' discussions and research since its publication. One of its recommendations is “Standardized Terminology and Reporting”. Little has been published to date on this topic, although conversations and dialogs on the subject are ongoing. The upshot of this communication is to draw attention to the problem of one term in particular, perimortem, which may be only the proverbial “tip of the iceberg” in the lexicon‐related concerns of forensic scientists. Even if it is an isolated issue, it is one that reflects the need for a consensus on term use and definitions by interdisciplinary practitioners who are currently using the term haphazardly, to the confusion of colleagues and potentially finders‐of‐fact in the courts.     

Refreshing the Aged Latent Fingerprints with Ionizing Radiation Prior to the Cyanoacrylate Fuming Procedure: A Preliminary Study

One of the crucial factors determining the cyanoacrylate deposit quality over latent fingerprints appeared to be the extent of the humidity. This work focuses on the enhancement/refreshment of age‐degraded latent fingerprints by irradiating the samples with UV, X‐ray, or thermal neutrons prior to the cyanoacrylate (CA) fuming. Age degradation of latent fingerprints deposited on glass surfaces was examined through the decrease in the number of characteristic minutiae counts over time. A term “critical day” was introduced for the time at which the average number of identifiable minutiae definitions drops to one‐half. Fingerprints older than their “critical day” were exposed to either UV, X‐ray, or thermal neutrons. Identical reference samples were kept unexposed. All samples, both reference and irradiated, were developed during a single CA fuming procedure. Comparative latent fingerprint analysis showed that exposure to ionizing radiation enhances the CA fuming, yielding a 20‐30% increase in average minutiae count.     

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.     

Alcohol‐Induced Blackout as a Criminal Defense or Mitigating Factor: An Evidence‐Based Review and Admissibility as Scientific Evidence

Alcohol‐related amnesia—alcohol blackout—is a common claim of criminal defendants. The generally held belief is that during an alcohol blackout, other cognitive functioning is severely impaired or absent. The presentation of alcohol blackout as scientific evidence in court requires that the science meets legal reliability standards (Frye, FRE702/Daubert). To determine whether “alcohol blackout” meets these standards, an evidence‐based analysis of published scientific studies was conducted. A total of 26 empirical studies were identified including nine in which an alcohol blackout was induced and directly observed. No objective or scientific method to verify the presence of an alcoholic blackout while it is occurring or to confirm its presence retrospectively was identified. Only short‐term memory is impaired and other cognitive functions—planning, attention, and social skills—are not impaired. Alcoholic blackouts would not appear to meet standards for scientific evidence and should not be admissible.     

The mourning process and “living with the dead”: Two case reports and a review of the literature

The discovery of mummified bodies in domestic settings is not unusual in the medico‐legal context. It is often a marker of social isolation, even in our urban modern society, and usually occurs among elderly people living alone or in precarious conditions. However, bereaved subjects can sometimes be found managing their grief by deliberately keeping the corpses of their loved ones at home. Investigation of these atypical cases can be challenging and often requires a multidisciplinary effort by different forensic specialists. We report two cases of people who lived for several months with the mummified remains of a relative. In both cases, the judge ordered a forensic psychiatry assessment of the survivors’ competency and the reasons for this peculiar behavior, which is regarded as abnormal in our society. Case 1 describes a shared psychosis, which developed out of a condition of extreme seclusion of the entire family. Case 2 shows that even a mild personality disorder on which a series of traumatic events operates can trigger psychotic decompensation, causing extreme denial of the reality of death. The analysis of these cases contributes to our knowledge of the scantly studied phenomenon of “Living with the Dead” and raises questions about the psychopathology behind it. It is useful to identify subjects who are more prone to developing this “deviant” behavior, in order to distinguish people with mental illness from those who merely want to profit from the death of a loved one.     

A 2‐Year Study of Δ 9‐tetrahydrocannabinol Concentrations in Drivers: Examining Driving and Field Sobriety Test Performance,,

From November 1, 2010 through November 30, 2012, 1204 whole‐blood samples were confirmed to contain THC alone or in combination with other drugs out of nearly 5000 Orange County, California, drivers suspected of driving under the influence of drugs. The goal of this study was to examine police reports and drug recognition expert evaluations of THC‐positive samples within this 2‐year time frame to determine whether there is a correlation between whole‐blood THC concentrations and field sobriety tests performance on DRE and non‐DRE evaluations. The FSTs prove to be sensitive to impairment by marijuana although as suspected, the findings of this study did not find a correlation between performance on field sobriety tests and the concentration of THC tested in whole‐blood samples. Driving behaviors were also examined and found to be similar to those seen in alcohol impairment. Future studies examining DRE findings are needed to confirm the results.     

Bloodstains on Leather: Examination of False Negatives in Presumptive Test and Human Hemoglobin Test

Presumptive tests for blood are very simple and sensitive tests used in the search for evidence. They also provide initial information on the nature of stains. A second test can confirm their nature. However, these tests can present false–negative results for different reasons. Some of those reasons have been studied, while others, those caused by the substrate material that contains the stain, are less well known. This work studies the effect of one component of a leather substrate—quebracho extract—on presumptive and human hemoglobin blood tests. Assays were performed using samples of blood dilutions contaminated with quebracho extract and others formed on a substrate containing the contaminant. Results show an undoubted interference that causes false negatives and even visible to the naked eye stains and also indicate that some tests (phenolphthalein) are more affected than others. Examiners should be taken into account when working on this kind of substrates.     

Effect of blood group active micro-organisms on the ABO grouping of human whole saliva

Three saliva samples with false positive ABO grouping results were assayed for blood group active organisms, using a variety of selective media to isolate representative strains from the salivary microflora. Eight out of 40, 8 out of 40 and 4 out of 30 strains from the three samples, respectively, showed blood group activity, which correlated well with the false positive specificities of the saliva samples. In all cases the false reaction only lasted a few days. Investigation of one of these samples before and after the appearance of the false positive activity yielded only one out of 40 blood group active organisms, using the same methods. Similar investigation of two "normal" saliva samples found none out of 40 and one out of 40 blood group active organisms, respectively. It is concluded that occasional false positive ABO grouping reactions of saliva samples are probably caused by the presence of unusually high numbers of blood group active micro-organisms, due to disturbances in the ecological balance of the salivary microflora.     

Validation of Half‐Reaction Amplification Using Promega PowerPlex® 16*

Abstract: DNA amplification is a fundamental yet costly process used in DNA analysis. This study evaluated half‐reaction amplification (12.5, 12, and 13 uL) using the Promega Powerplex^® 16 Kit with the hope of reducing sample analysis costs by half. A sensitivity study was completed, along with the testing of various blood stain samples including those with low (<0.40 ng) and high DNA concentrations (>3.0 ng), peak height imbalances, and allelic drop‐out. Also, 467 samples submitted to the MUFSC laboratory for testing were analyzed. Results indicate that half‐reaction amplification produced higher quality profiles than full‐reactions. Average peak heights increased by 85%, peak height imbalances improved, and drop‐out was eliminated in 75.8% of samples. Only eight of 467 case samples required re‐amplification, a success rate of 94% was observed, and the repeat rate decreased significantly. Finally, a DNA input of 0.25–1.0 ng is ideal for half‐reaction amplification.     

Mitochondrial DNA Extraction from Burial Soil Samples at Incremental Distances: A Preliminary Study

Preservation variance of soil DNA is neglected in the literature, and exceptional cases exaggerate amplification capabilities. This study sought to amplify a short mitochondrial fragment (212 bp) specific to Sus scrofa domesticus from the soil surrounding decomposing pig remains from an open‐air locale. Samples collected above the body at incremental distances after 145 days of initial placement yielded pig DNA. A secondary sampling was collected in 2017, approximately 768 days after burial. Inhibition tests corroborated that pig DNA was no longer present in the soil resulting in a loss of original DNA between 145 and 768 days. The results provide evidence that genetic material leaches out radially from the source and DNA fragments longer than 200 bp do not persist in soil for a relatively short timeframe in western Montana. The conclusions support the collection of soil in crime scene investigation procedures within the first few months of decomposition.     

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.     

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real‐time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples.     

Delirium Due to Intoxication from the Novel Synthetic Tryptamine 5‐MeO‐DALT

Synthetic tryptamines have gained popularity for their hallucinogenic properties, unscheduled status, and availability from “head shops” and through the internet. Here, we present a case of synthetic tryptamine‐induced delirium secondary to 5‐MeO‐DALT ingestion in a previously healthy young male. 5‐MeO‐DALT led to the hospitalization of our patient after ingestion of a standard dose, presenting with extreme agitation, tachycardia, diaphoresis, and combativeness leading to physical restraint and intravenous sedation. A search of PubMed, Ovid, and Google Scholar for keywords of “5‐MeO‐DALT,” “5‐methoxy‐N,N‐diallyltryptamine,” or “Lucy‐N‐Nate” found no case reports or clinical articles in the literature. Rapid emergence and commercialization of this novel synthetic tryptamine 5‐MeO‐DALT points to the importance of health care and forensic professionals keeping abreast of the latest drugs of abuse and their clinical features. The authors hope this report leads the way in disseminating the potential risks associated with unscheduled and unregulated substances, synthetic tryptamines such as 5‐MeO‐DALT in particular.