Validation of the AmpF?STR SEfiler Plus™ PCR Amplification kit for forensic STR analysis

Validation of the AmpF?STR^® SEfiler Plus™ PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpF?STR^® SGM Plus™ kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but showed more stutters, drop-ins, drop-outs and allelic imbalances.     

Developmental validation of RSID™-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen

Abstract: Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate‐specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID?‐Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID?‐Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross‐react with other human or nonhuman tissues tested. RSID?‐Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA‐based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID?‐Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.     

The importance of dark adaptation for forensic examinations; an evaluation of the Crime-lite Eye™

Forensic practitioners are recommended to dark adapt their eyes prior to conducting evidential searches in the dark. The dark adaptation process remains poorly standardised across the discipline, with little quantified regarding the benefits of such preparative steps. Herein, we report the findings of a study that recruited 50 participants to assess the effectiveness of the Crime-lite Eye?, a darkness adaptation device developed to assist forensic practitioners both in the laboratory and in field. Participants were tasked with searching for the fluorescent signatures left by reaction of 1,8-diazafluoren-9-one (DFO) with amino acids, in a manner akin to the fluorogenic fingerprint treatment of porous evidence. Using an Epson Stylus Photo R265 inkjet printer, ink cartridges were filled with alanine solutions of various concentrations, allowing different motifs to be printed onto copy paper and subsequently developed using DFO. Participants searched for this ‘evidence’ both with and without dark adapted vision. On average, participants were able to locate and correctly recognise 16% more evidence once dark adapted using the Crime-lite Eye?.The increase in evidence located by participants once dark adapted suggests that crime scene officers should be dark adapting in order to visualise as much as possible. The time taken to dark adapt, 10?min on average during this study, is not excessively long, and should not significantly slow the investigation.     

Developmental validation of the SPERM HY-LITER™ kit for the identification of human spermatozoa in forensic samples

Abstract: With sexual assault evidence, the visualization of spermatozoa confirms that ejaculation has occurred. However, microscopic examination of spermatozoa is a laborious process and can sometimes result in sperm cells being overlooked. Here, we present the developmental validation of the SPERM HY‐LITER? kit, which contains a human sperm–specific mouse monoclonal antibody coupled to a fluorescent Alexa 488 dye. The kit was tested using samples of human semen, saliva, blood, and urine, various animal semen extracts, sexual lubricants, and a commercially available spermicidal film. Postcoital vaginal swabs, degraded semen samples, and samples prepared with sample fixation techniques that deviated from the kit‐provided protocol were also tested. In each case, the SPERM HY‐LITER? kit was demonstrated to bind only to human sperm cell heads. Limitations to this fluorescent staining procedure include nonspecific staining and increased background fluorescence with extreme heat fixation in some samples.     

Rapid screening for the detection and differentiation of γ-hydroxybutyrate using ion chromatography

The analysis of gamma-hydroxybutyric acid (GHB) is problematic because it is hygroscopic, it lacks a good UV chromophore, and it undergoes heat-induced cyclization. This paper presents a new method utilizing ion-exchange chromatography (IC) with conductivity detection. The simple sample preparation, rapid analysis time, and inorganic anion detection capabilities are all advantages over the current methods. The detection of inorganic salts (formed during GHB synthesis) gives insight into the synthetic route utilized and can aid in drug seizure comparison. The developed method has a detection limit for GHB anions of 0.57 mg/L and chloride of 0.22 mg/L. A comparison of this technique with a current gas chromatography-mass spectrometry technique is presented, and a t-test found that the two methods' results are not statistically different at the 99.9% confidence level demonstrating the merits of this fast, simple, and informative IC method as a routine screening tool.     

Validation study of KPICS SpermFinder™ by NicheVision Forensics, LLC for the identification of human spermatozoa

Abstract: Microscopic analysis for the identification of spermatozoa is commonly performed during the forensic examination of sexual assault evidence. Two widely utilized methods for the confirmation of the presence of spermatozoa are visualization of the cells via phase‐contrast microscopy with wet mounted samples and bright field microscopy with histologically stained samples. The KPICS SpermFinder? by NicheVision Forensics, LLC accelerates this time‐consuming process via an automated microscope with an algorithm designed to locate spermatozoa on a Christmas tree histologically stained microscope slide. Upon a qualified scientist’s review of the generated data, the KPICS SpermFinder? was able to locate spermatozoa, typically finding on average 106.28% ± 115.37% more spermatozoa than with manual examinations. The KPICS SpermFinder? provided the location of identified cells with reproducible results.     

Validation of the Seratec® SeraQuant™ for the quantitation of prostate-specific antigen levels on immunochromatographic membranes

Abstract: Use of immunochromatographic membranes for the detection of prostate‐specific antigen (PSA) has become commonplace in forensic laboratories. Experiments were designed to test the newly developed Seratec^® SeraQuant? for accuracy, precision, and consistency in the quantitation of PSA. PSA standards were diluted with buffers and run on the instruments. Values obtained were examined for accuracy (was the correct value obtained?) and precision (were multiple sample values consistent?). To test for variation between instruments, large volumes of diluted PSA standard were run repeatedly on six units and the values obtained were plotted against the known PSA values to obtain a standard curve for each instrument. Fifty membranes having negative or weak positive results were then run on the six units, and the adjusted values were recorded and compared. Results of these experiments indicate that the instruments are accurate and precise in the quantitation of low levels of PSA.     

AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples

Abstract: The AutoMate Express? Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler? lysis buffer or PrepFiler BTA? lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep? column, the lysate in the sample tubes were loaded onto AutoMate Express? instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework‐type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day‐to‐day operation.     

Validation of the InnoXtract™ DNA extraction method for bone,teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.     

GeneMarker® HID: A reliable software tool for the analysis of forensic STR data

Abstract: GeneMarker^® HID was assessed as a software tool for the analysis of forensic short tandem repeat (STR) data and as a resource for analysis of custom STR multiplexes. The software is easy to learn and use, and includes design features that have the potential to reduce user fatigue. To illustrate reliability and accuracy, STR data from both single‐source and mixture profiles were analyzed and compared to profiles interpreted with another software package. A total of 1898 STR profiles representing 28,470 loci and more than 42,000 alleles were analyzed with 100% concordance. GeneMarker HID was also used to successfully analyze data generated from a custom STR multiplex, with simplified and rapid implementation. Finally, the impact of the user‐friendly design features of the software was assessed through a time scale study. The results suggest that laboratories can reduce the time required for data analysis by at least 25% when using GeneMarker HID.     

Validation studies in forensic odontology – Part 1: Accuracy of radiographic matching

As part of a series of studies aimed at validating techniques in forensic odontology, this study aimed to validate the accuracy of ante-mortem (AM)/postmortem (PM) radiographic matching by dentists and forensic odontologists. This study used a web-based interface with 50 pairs of AM and PM radiographs from real casework, at varying degrees of difficulty. Participants were shown both radiographs as a pair and initially asked to decide if they represented the same individual using a yes/no binary choice forced-decision. Participants were asked to assess their level of confidence in their decision, and to make a conclusion using one of the ABFO (American Board of Forensic Odontology), INTERPOL (International Criminal Police Organisation) and DVISys? (DVI System International, Plass Data Software) identification scale degrees. The mean false-positive rate using the binary choice scale was 12%. Overall accuracy was 89% using this model, however, 13% of participants scored below 80%. Only 25% of participants accurately answered yes or no > 90% of the time, with no individual making the correct yes/no decision for all 50 pairs of radiographs. Non-odontologists (lay participants) scored poorly, with a mean accuracy of only 60%. Use of the graded ABFO, DVISYS and INTERPOL scales resulted in general improvements in performance, with the false-positive and false-negative rates falling to approximately 2% overall. Inter-examiner agreement in assigning scale degrees was good (ICC = 0.64), however there was little correlation between confidence and both accuracy or agreement among practitioners. These results suggest that use of a non-binary scale is supported over a match/non-match call as it reduces the frequency of false positives and negatives. The use of the terms “possible” and “insufficient information” in the same scale appears to create confusion, reducing inter-examiner agreement. The lack of agreement between higher-performing and lower-performing groups suggests that there is an inconsistency in the cognitive processes used to determine similarity between radiographs.     

Practical benzylation of N,N-substituted ethanolamines related to chemical warfare agents for analysis and detection by electron ionization gas chromatography–mass spectrometry

The benzylation of three low molecular weight N,N-disubstituted ethanolamines related to chemical warfare agents (CWAs) to furnish derivatives with improved gas chromatography-mass spectrometry (GC-MS) profiles is described. Due to their low molecular weight and polar nature, N,N-disubstituted ethanolamines are notoriously difficult to detect by routine GC-MS analyses during Organisation for the Prohibition of Chemical Weapons (OPCW) proficiency tests (PTs), particularly in scenarios when they are present at low levels (~1–10 ppm) amidst more abundant interferences. Our studies revealed that the optimal derivatization conditions involved the treatment of the ethanolamine with benzyl bromide in the presence of an inorganic base (e.g., Na₂CO₃) in dichloromethane at 55°C for 2 h. This optimized set of conditions was then successfully applied to the derivatization of N,N-dimethylethanolamine, N,N-diethylethanolamine and N,N-diisopropylethanolamine present separately at 1 and 10 μg/mL concentrations in a glycerol-rich matrix sample featured in the 48th OPCW PT. The benzylated derivatives of the three ethanolamines possessed retention times long enough to clear the massive glycerol-containing matrix interferences. The protocol herein is introduced as an alternative method for derivatization of these CWA and pharmaceutically important species and should find broad applicability in laboratories where routine forensic analysis is carried out.     

The ‘relics of Joan of Arc’: A forensic multidisciplinary analysis

Archaeological remains can provide concrete cases, making it possible to develop, refine or validate medico-legal techniques.In the case of the so-called ‘Joan of Arc's relics’ (a group of bone and archaeological remains known as the ‘Bottle of Chinon’), 14 specialists analysed the samples such as a cadaver X of carbonised aspect: forensic anthropologist, medical examiners, pathologists, geneticists, radiologist, biochemists, palynologists, zoologist and archaeologist. Materials, methods and results of this study are presented here.This study aims to offer an exploitable methodology for the modern medico-legal cases of small quantities of human bones of carbonised aspect.     

Developmental validation of the Microreader™ Y Prime Plus ID System: An advanced Y-STR 38-plex system for forensic applications

Y-STR is widely used in sexual assaults and familial searches of suspects. Here, we reported a novel 38-plex STR genotyping system designed for forensic applications. Microreader? Y Prime Plus ID System (YPP) amplifies 38 loci in one reaction, including 29 loci from commonly used Yfiler® Plus PCR Amplification Kit & PowerPlex® Y23 System (DYS393, DYS570, DYS19, DYS392, DYS549, Y GATA H4, DYS460, DYS458, DYS481, DYS635, DYS448, DYS533, DYS449, DYS456, DYS389I, DYS390, DYS389Ⅱ, DYS438, DYS391, DYS439, DYS437, DYS385a/b, DYS643, DYS518, DYS576, DYF387S1a/b, and DYS627), 6 commonly used loci for the Y-STR database (DYS444, DYS447, DYS596, DYF404a/b, DYS527a/b, DYS557) and one Y-indel specific for the Chinese population. YPP is designed for different types of samples, such as blood card and swabs. In this work, YPP was validated following SWGDAM guidelines (2016) and guidelines from Ministry of Public Security of the People’s Republic of China, including PCR-based, sensitivity, accuracy and precision, mixture, stability and inhibitor, and species specificity. The results indicate that the Microreader? Y Prime Plus ID System is a powerful identification kit designed for forensic databases.     

Examples of kinship analysis where Profiler Plus™ was not discriminatory enough for the identification of victims using DNA identification

The identification of the victims of the 2009 Victorian bushfires disaster, as in other mass disasters, relied on a number of scientific disciplines - including DNA analysis. As part of the DVI response, DNA analysis was performed to assist in the identification of victims through kinship (familial matching to relatives) or direct (self source of sample) matching of DNA profiles. The majority of the DNA identifications made (82%) were achieved through kinship matching of familial reference samples to post mortem (PM) samples obtained from the victims. Although each location affected by the bushfires could be treated as a mini-disaster (having a small closed-set of victims), with many such sites spread over vast areas, DNA analysis requires that the short tandem repeat (STR) system used be able to afford enough discrimination between all the DVI cases to assign a match. This publication highlights that although a 9-loci multiplex was sufficient for a DVI of this nature, there were instances that brought to light the short comings of using a 9-loci multiplex for kinship matching--particularly where multiple family members are victims. Moreso it serves to reinforce the recommendation that a minimum of 12 autosomal STR markers (plus Amelogenin) be used for DNA identification of victims which relies heavily on kinship matching.     

A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30–150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%–75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.     

What do prosecutors maximize? An analysis of the federalization of drug crimes

Recent legislation has expanded the jurisdiction of the federalgovernment over crimes that were traditionally prohibited onlyby state law. We model the decision-making process of stateand federal prosecutors, and the determinants of prosecutors'decisions to allocate drug cases to the state versus the federalsystems. Using 1991 surveys of state and federal inmates incarceratedfor drug crimes, we find that individuals who hire private attorneysand who are high-human-capital and successful in the legitimatesector are more likely to end up in the federal system. Thisis consistent with the model in which prosecutors maximize boththe payoffs from eliminating crime and their private human capital.     

A hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry method for the simultaneous determination of γ-hydroxybutyrate and its precursors in forensic whole blood

A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of γ-hydroxybutyric acid (GHB), γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in human ante-mortem and post-mortem whole blood. The blood proteins were precipitated using a mixture of methanol and acetonitrile, and the extract was cleaned-up by passage through a polymeric strong cation exchange sorbent. Separation of the analytes and their structural isomers was obtained using a column with a zwitterionic stationary phase. Matrix-matched calibrants, combined with isotope dilution, were used for quantitative analysis. GHB was determined in both positive and negative ion modes. The relative intra-laboratory reproducibility standard deviations were better than 10% and 6% for blood samples at concentrations of 2mg/L and 20-150mg/L, respectively. The mean true extraction recoveries were 80% for GHB and greater than 90% for GBL and 1,4-BD at concentration levels of 20-50mg/L. The limits of detection were approximately 0.5mg/L for GHB and GBL, and 0.02mg/L for 1,4-BD in ante-mortem blood. The corresponding lower limits of quantification were less than 1mg/L for GHB and GBL, and less than 0.1mg/L for 1,4-BD. GBL was unstable in whole blood freshly preserved with a sodium fluoride oxalate mixture, but the stability could be improved significantly by preservation with a sodium fluoride citrate EDTA mixture.     

Hair analysis for drugs of abuse. Hair color and race differentials or systematic differences in drug preferences?

There is currently a debate in the literature on chemical drug analysis concerning the contribution of biophysical attributes associated with specimens and specimen donors to assay outcome. In recent years this debate has focused on hair analysis, but has in the past also been raised in urinalysis interpretation. In this article we examine several aspects of that controversy. First, we present data regarding the effects of hair color on the distribution of positive hair testing results for three drug classes. We compare these results to negative hair samples from comparable donors. This data is derived from head hair from preemployment donors that was classified according to seven visual color categories. We determined the distribution of colors for hair samples devoid of any of three assayed drugs (amphetamines, cocaine, and cannabinoids). Subsequently, this distribution was compared with the distributions for hairs that had tested positive for amphetamines, cocaine or cannabinoids. We examined a total of 2000 randomly selected samples; 500 negative hair samples and 500 positive samples for each of three drugs: cannabinoids, cocaine, and amphetamine. We also evaluated ethnic/racial factors in relation to positive urinalyses for various ethnic/racial groups. We examined approximately 4000 urine specimens from two different groups, each constituting around 2000 specimens. In addition to ethnicity/race and urinalysis outcome, we also examined the relationship between the hair color distributions of urine donors and the corresponding urinalysis results for the three drug classes. We also compared them to drug-negative samples. Our summary impression is that the observed outcome patterns were largely consistent with differences in drug preferences among the various societal groups. There was little evidence of a pattern attributable to hair color bias alone or selective binding of drugs to hair of a particular color. Likewise, there was no discernible pattern associated with race or ethnicity that would lend support to a "race effect" in drug analysis.