Evaluation of Bluestar® Forensic Magnum and Other Traditional Blood Detection Methods on Bloodstained Wood Subjected to a Variety of Burn Conditions.

Accurate blood detection is a primary concern for forensic scientists, especially in highly compromised situations. In this study, blood was added to wood blocks and subjected to a variety of fire treatments: the absence or presence of accelerant, burn time (1, 3, or 5 min), and extinguishment method (smothering or dousing with water). Burned blocks were given a qualitative burn score, followed by removal of half of the char from each block and subsequent testing of each half for blood using luminol (13% positive; n = 96), Bluestar® Forensic Magnum (5.2% positive; n = 96), and combined phenolphthalein tetramethylbenzidine test (0% positive; n = 192). Luminol and Bluestar® Forensic Magnum performed similarly, both outperforming PTMB. Additionally, positive results were more likely from samples that were smothered, had a low burn score, and had more concentrated blood solutions (neat or 1:2). Overall, it is extremely unlikely that blood would be detected on combustible substrates exposed to direct fire.

Rapid differentiation of epithelial cell types in aged biological samples using autofluorescence and morphological signatures.

Establishing the tissue source of epithelial cells within a biological sample is an important capability for forensic laboratories. In this study we used Imaging Flow Cytometry (IFC) to analyze individual cells recovered from buccal, epidermal, and vaginal samples that had been dried between 24 hours and more than eight weeks. Measurements capturing the size, shape, and fluorescent properties of cells were collected in an automated manner and then used to build a multivariate statistical framework for differentiating cells based on tissue type. Results showed that epidermal cells could be distinguished from vaginal and buccal cells using a discriminant function analysis of IFC measurements with an average classification accuracy of ~94%. Ultimately, cellular measurements such as these, which can be obtained non-destructively, may provide probative information for many types of biological samples and complement results from standard genetic profiling techniques.

Development of a normalized extraction to further aid in fast, high-throughput processing of forensic DNA reference samples.

The goal of this project was to develop a "normalized" extraction procedure to be used in conjunction with previously validated 3µL fast PCR reactions (42-51min utilizing KAPA2G™ Fast Multiplex PCR Kit) and alternative capillary electrophoresis (24-28min injection using POP-6™ Polymer and a 22cm array). This was the final phase of a workflow overhaul for the database unit at Cellmark Forensics to achieve a substantial reduction in processing time for forensic DNA database samples without incurring significant added costs and/or the need for new instrumentation, while still generating high quality STR profiles. Extraction normalization aimed to consistently yield a small range of DNA concentrations, thereby eliminating the need for sample quantification and dilution. This was specifically achieved using the ChargeSwitch® Forensic DNA Purification Kit and a reduction in extraction bead quantity, thereby forcing an increase in bead binding efficiency. Following development of this extraction procedure, an evaluation ensued to assess the combination of normalized extraction, 3µL fast PCR (with PowerPlex 16 HS, Identifiler Plus and Identifiler primer sets), and alternative CE detection - further referred to as new "first pass" procedures. These modifications resulted in a 37% reduction in processing time and were evaluated via an in depth validation, from which nearly 2000 STR profiles were generated, of which 554 profiles from 77 swab donors and 210 profiles from 35 buccal collector donors specifically arose from the new first pass procedures. This validation demonstrates the robustness of these processes for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated and their ability to generate high quality STR profiles with 95-99% and 88-91% pass rates, respectively.

Validation of alternative capillary electrophoresis detection of STRs using POP-6 polymer and a 22cm array on a 3130xl genetic analyzer.

The goal of this project was to reduce capillary electrophoresis detection time on a 3130xl Genetic Analyzer for amplification product obtained from 4-dye and 5-dye STR amplification kits while still generating high quality STR profiles. This was accomplished by utilizing a more viscous polymer (POP-6™) and a shorter array (22 cm) than that which are typically used (POP-4(®) polymer and a 36 cm array) for human identification purposes. Spatial calibration and detection run modules were modified in response to the use of this polymer/array combination and to reduce detection time. Alternative detection resulted in 24-28 min run times, as compared to ∼45 min using traditional POP-4(®)/36 cm detection methods. POP-6™/22 cm detection run modules were validated for use with 4-dye Promega STR kits (e.g., PowerPlex(®) 16 and PowerPlex(®) 16HS) and 5-dye Life Technologies kits (e.g., Identifiler(®) and Identifiler(®) Plus). Three hundred ninety-five samples, controls and allelic ladders were used for the validation studies, which consisted of a comparison of alternative POP-6™/22 cm detection to traditional POP-4(®)/36 cm (including reproducibility/concordance of allele calls, resolution, ILS sizing quality, peak height and pass rates), a sizing study (precision and accuracy) and a sensitivity study to obtain a usable range of injection times. Compared to traditional POP-4(®)/36 cm detection, alternative detection resulted in 100% reproducible and concordant alleles, the ability to achieve one base resolution, slightly reduced ILS sizing quality, slightly reduced peak height and statistically similar pass rates (α=0.05). It should be noted that alternative detection offered improved resolution over that of traditional for amplicons less than ∼200 b, but had reduced resolution for products greater than ∼200 b. Additionally, alternative detection yielded acceptable precision and accuracy of sizing using Life Technologies criteria (<0.15 standard deviation of allele sizing and ±0.5b sizing differences for the same allele) and usable injection parameters of 2 kV 4-15s (compared to 3 kV 10s for traditional). The run modules developed and validated for 4-dye and 5-dye STR kits using POP-6™ polymer on a 22 cm array offer a tremendous reduction in detection time (∼40%) while still generating high quality STR profiles.