Elemental and molecular characterization of degrading blood pools.

Blood is a commonly encountered type of biological evidence and can provide critical information about the crime that occurred. The ability to accurately and precisely determine the time since deposition (TSD) of a bloodstain is highly sought after in the field of forensic science. Current spectral methods for determining TSD are typically developed using small volume bloodstains, we investigate the applicability to larger volume blood pools where drying and degradation mechanics are different. We explored the differences that exist between the surface and bulk of dried segments from fragments collected from 15 mL dried blood pools and identified heterogeneity using RGB colour analysis and hierarchical cluster analysis (HCA). The physical, molecular, and atomic differences between the layers were further investigated using scanning electron microscopy (SEM), X-Ray photoelectron spectroscopy (XPS), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and Raman spectroscopy. SEM identified different morphology on the surface and the bulk indicative of density-dependant cellular settling. XPS revealed that iron was not present on the surface but rather was present in the bulk where the red blood cells had settled. The oxidation state of the iron was quantified over three weeks in which it transitioned from entirely Fe2+ to primarily Fe3+, as expected for ex vivo degradation of hemoglobin. Further, indications of amide saponification occurring at the blood-air interface were identified in the increased quantity of the C-O moiety relative to CO, and the formation of free amines and OC-ONa groups over time. ATR-FTIR and Raman spectroscopy provided insights into differences in the molecular composition of the layers, suggesting that the surface consists of more nucleic acids, lipids, and glycoproteins than the bulk, which was dominated by proteins (p < 0.001% using principal component analysis (PCA)). Additionally, spectral band trends previously reported to have applicability to the estimation of TSD were observed for the bulk portion of the blood pool as the Hb underwent predictable time dependant changes from oxyHb to metHb. PCA was performed based on all spectral data which demonstrated statistically significant differences between the surface and bulk, as well as proof-of-concept for linear TSD estimation models.

Optical profilometry for forensic bloodstain imaging.

Understanding the physical, chemical and biological changes that occur during the drying of a bloodstain is important in many aspects of forensic science including bloodstain pattern analysis and time since deposition estimation. This research assesses the use of optical profilometry to analyze changes in the surface morphology of degrading bloodstains created using three different volumes (4, 11, and 20 µL) up to 4 weeks after deposition. We analyzed six surface characteristics, including surface average roughness, kurtosis, skewness, maximum height, number of cracks and pits, and height distributions from the topographical scans obtained from bloodstains. Full and partial optical profiles were obtained to examine long-term (minimum of 1.5-h intervals) and short-term (5-min intervals) changes. The majority of the changes in surface characteristics occurred within the first 35 min after bloodstain deposition, in agreement with current research in bloodstain drying. Optical profilometry is a nondestructive and efficient method to obtain surface profiles of bloodstains, and can be integrated easily into additional research workflows including but not limited to time since deposition estimation. Optical profilometry is a non-contact tool to scan bloodstains in ambient conditions Drying phases are observable in small drip bloodstains Significant surface morphology changes occur within 35 min after deposition.

Alginate/xanthan gum hydrogels as forensic blood substitutes for bloodstain formation and analysis.

Understanding the behaviour of human blood outside of the body has important implications in forensic research, especially related to bloodstain pattern analysis (BPA). The design of forensic blood substitutes (FBSs) can provide many advantages, including the incorporation of multiple physiological components for use as safe and reliable materials for forensic applications. In this work, we present the design of synthetic alginate and xanthan gum-based hydrogels that contain electrosprayed microparticles (MPs) with and without crosslinked DNA. In addition to the MPs, the alginate/xanthan gum FBS materials include fillers to alter the physical appearance and fluid properties of the material. The optimized FBS consisted of alginate (1% w/v) and xanthan gum (5.0 × 10-3% w/v), 2 mM CaCl2, ferric citrate (0.5% w/v), magnesium silicate (0.25% w/v), Allura Red dye (2% w/v), 0.025% v/v Tween 20 and 9.5% v/v MPs. The FBS was tested in passive dripping experiments relevant to BPA scenarios at various impact angles. The spreading ratio (Ds/D0) was determined for 90° stains made on a paper surface and compared to bovine blood where the FBS was shown to simulate accurate and predictable spreading behaviour. In addition, we simulated other common BPA scenarios (e.g., impact patterns) and evidence processing potential. The FBS could be swabbed, and the DNA could be extracted, amplified, and genotyped analogous to human blood evidence. A stability test was also conducted which revealed a shelf-life of over 4 weeks where the material remains relevant to human blood at physiological temperature.

Examination of Bovine Red Blood Cell Death in Vitro in Response to Pathophysiologic Proapoptotic Stimuli.

BACKGROUND: Interspecies variations in mammalian red blood cells (RBCs) are observed in circulating RBC lifespan, cell size, fluidity, aggregation, water permeability, metabolism, lipid composition, and the overall proteome. Bovine RBC cell membrane is deficient in phosphatidylcholine and exhibits anomalies in the arrangement of phosphatidylethanolamine within the lipid bilayer. However, like human RBCs, virtually all the aminophospholipid phosphatidylserine (PS) is found within the cytoplasmic side of the cell membrane of intact circulating bovine RBCs. During apoptotic cell death of human and murine RBCs, PS translocates to the outer leaflet of the cell membrane via Ca2+-dependent and -independent signaling mechanisms. However, little is known about this process in bovine RBCs. METHODS: Using cytofluorometry analyses, we characterized and compared the cell death responses in bovine and human RBCs in vitro exposed to various pathophysiologic cell stressors. RESULTS: Ionic stress, by ionophore treatment, and oxidative stress enhanced cytoplasmic Ca2+ levels and cell membrane PS expression in both bovine and human RBCs. Fever-grade hyperthermia and energy starvation promoted Ca2+ influx and elevated reactive oxygen species levels in both human and bovine RBCs. However, bovine RBCs displayed minimal increases in PS expression elicited by hyperthermia, energy starvation, and extracellular hypertonicity as compared to human RBCs. In response to decreased extracellular osmolality, bovine RBCs exhibited significantly enhanced fragility as compared to human RBCs. CONCLUSIONS: Bovine RBCs display differential cell death patterns as compared to human RBCs, only partly explained by increased Ca2+ influx and oxidative stress. Premature removal of circulating RBCs could potentially contribute to the pathogenesis of anemia in cattle caused by a wide range of factors such as systemic diseases, parasitic infections, and nutritional deficiencies.

Using total RNA quality metrics for time since deposition estimates in degrading bloodstains.

The physicochemical changes occurring in biomolecules in degrading bloodstains can be used to approximate the time since deposition (TSD) of bloodstains. This would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has traditionally targeted mRNA and miRNA. Bovine blood with ACD-A anticoagulant was deposited and stored in plastic microcentrifuge tubes at 21 or 4°C and tested over different timepoints spanning 1 week. Total RNA was extracted from each sample and analyzed using automated high sensitivity gel electrophoresis. Nine RNA metrics were visually assessed and quantified using linear and mixed models. The RNA Integrity Number equivalent (RINe) and DV200 were not influenced by the addition of anticoagulant and demonstrated strong negative trends over time. The RINe model fit was high (R2  = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200. This suggests that these standardized metrics can be directly compared between scenarios and individuals, with DV200 having an inflection point at approximately 28 h. This study provides a novel approach for blood TSD research, revealing metrics that are not affected by inter-individual variation, and improving our understanding of the rapid RNA degradation occurring in bloodstains.

Calcium-Alginate Tissue Gels (CATG): Proof-of-concept biomaterial development.

Hydrogels are desirable materials to the field of forensic science and offer many advantages for use as tissue simulants in research and training scenarios. In this work, we demonstrate a proof-of-concept study for our biomaterial described as the Calcium-Alginate Tissue Gel (CATG). CATG biomaterials integrate functional DNA strands designed to amplify with known human primer sets for genetic profiling. Our range of CATG materials demonstrate successful DNA extraction, PCR amplification and genotyping when both fresh and aged for 21 days. The rheological properties of the CATGs were measured and the incorporation of DNA into the CATGs was assessed. Overall, the CATGs demonstrated increased viscoelastic behavior with the addition of DNA. In addition, two methods of sampling were considered, where it was found that cutting a sample of the dried CATG produced higher allele peak heights in the genotype compared to swabbing. Overall, our CATG biomaterials can be designed for multiple applications in forensic science with tunable properties for various training and research needs.

Whole bovine blood use in forensic research: Sample preparation and storage considerations.

Mammalian whole blood sources are often used for forensic research and training when human samples cannot be sourced. While porcine, ovine and equine blood have been shown to be viable alternatives to whole human blood for forensic purposes, procurement can still pose a problem, especially for smaller and remote institutions. This work explores the use of whole bovine blood for basic bloodstain simulation. Sample preparation through the addition of ACD-A anticoagulant was optimized and storability was explored. Viscosity, surface tension, density, and packed cell volume, four fluid properties relevant to bloodstain pattern analysis, were monitored over four days and in two temperature conditions. Linear mixed models accounting for variation in the donor demonstrated that these fluid properties of the bovine blood changed predictably over time and with temperature. Whole bovine blood with 12.5% v/v ACD-A was found to be viable for use in basic bloodstain simulation at ambient and physiological temperature.

Untargeted SPME-GC-MS Characterization of VOCs Released from Spray Paint.

Paints are a common form of physical evidence encountered at crime scenes. This research presents an optimized method for the untargeted analysis of volatile organic compounds (VOCs) in spray paint using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). The presence and persistence of VOCs were monitored in 30 minute intervals, over a 4 hour period, in a triplicate time study. As predicted, spray paint solvents are lost to the environment readily, whereas few VOCs remained present in the headspace in low concentrations beyond 4 hours. The VOCs that were observed to have the highest persistence in the headspace were aromatic compounds and those with longer hydrocarbon chains. We present this study in a forensic science context and suggest that the interpretation of the results may be useful for forensic applications in establishing a time since deposition of a spray-painted surface.

Quantifying visible absorbance changes and DNA degradation in aging bloodstains under extreme temperatures.

Physicochemical property changes observed in a degrading bloodstain can be used to estimate its time since deposition (TSD) and provide a timestamp to the sample's age. Many of the time-dependent processes that occur as a bloodstain degrades, such as DNA fragmentation and changes in hemoglobin structure, also exhibit temperature-dependent behaviours. Previous studies have demonstrated that pairing high-resolution automated gel electrophoresis and visible absorbance spectroscopy could be used to quantify the rate of degradation of a bloodstain in relation to time and storage substrate. Our study investigates such trends with an added factor, extreme temperatures. Passive drip stains were stored in either microcentrifuge tubes or on FTA cards at either -20°C, 21°C or 40°C and tested over 11 time points spanning 15 days. For both storage substrates, the wavelength at maximum absorbance for the Soret band and the maximum absorbance of the Alpha band showed a negative trend over time suggesting that spectral shifts are informative for TSD estimates. The ratio of the maximum peak height for DNA fragments lengths of 500-1000 base pairs to 1000-5000 base pairs was the most informative DNA variable in relation to time for both substrates. Cross-validation suggested the appropriate fit of the models with the data and reasonable predictive ability. We integrated both DNA concentration and hemoglobin visible absorbance metrics using principal component analysis (PCA) into a single model. Adding the random effect of the donor to the PCA model accounted for a large portion of the variation as did storage method and temperature. Additionally, canonical correspondence showed that temperature corresponded differently to the response variables for FTA card and microcentrifuge tube samples, suggesting a substrate specific effect. This study confirms that pairing DNA concentration and hemoglobin's visible absorbance can provide insight on the effect of different environmental and storage conditions on bloodstain degradation. While the level of uncertainty surrounding TSD estimates still precludes its use in the field, this study provides a valuable framework that improves our understanding of variation surrounding TSD estimates, which will be critical to any eventual application.

Characterizing drip patterns in bloodstain pattern analysis: An investigation of the influence of droplet impact velocity and number of droplets on static pattern features.

This work characterizes fundamental features of static drip patterns simulated for forensic bloodstain pattern analysis. The purpose of this study was to determine if and how two independent variables, impact velocity and droplet number, influence the parent stain size, shape and satellite spatter distribution of drip patterns created with whole ovine blood. To do this, 500 drip patterns were created in a controlled environment at five varying impact velocities and ten different droplet numbers on a hard paper surface. Digital images of the dried patterns were processed and analyzed using Fiji (ImageJ). The data collected from each pattern support the hypotheses that drip patterns contain predictable and reproducible elements based on impact velocity and droplet number. Basic fluid dynamic principles demonstrate that the size of the parent stains, as well as the number and distribution of satellite stains increase with increasing Weber number. A decrease in the circularity of the parent stains was also noted. This study provides fundamental qualitative and quantitative data on observable elements of drip patterns that can be used practically by bloodstain pattern analysts for pattern identification and classification.

Quantifying chemiluminescence of the forensic luminol test for ovine blood in a dilution and time series.

This study investigates the chemiluminescent reaction of whole ovine blood with a luminol solution in a time and dilution series. Replicate samples of both fresh and dried certified pathogen-free ovine blood were prepared and diluted. Seven dilution conditions from neat to 1:1000000 were created for testing. A luminol solution, created using the standard Weber protocol, was applied to all samples in controlled laboratory conditions. A SpectraMax® M3 microplate reader luminometer was used to quantify the chemiluminescence from the reactions as relative luminescence units (RLUs) every four seconds for three minutes. Trends within and amongst the times series, reaction half-lives, and maximum chemiluminescent intensities are discussed. Our research provides a comprehensive dataset derived from instrumental and visual observations on the chemiluminescence resulting from ovine blood's reaction to luminol. This study has implications in forensic bloodstain pattern analysis as it offers a mixed method approach to characterizing the reaction between blood and a commonly used presumptive testing reagent.

The application of silicon sol-gel technology to forensic blood substitute development: Investigation of the spreading dynamics onto a paper surface.

This work investigates the spreading dynamics of three candidate sol-gel solutions, of ranging viscosities, surface tensions and densities, and compares them with water and two commercial blood substitute products. Droplets were created with different sizes (10 to75µL) and impact velocities (1.4 to 6.0m/s) to strike 176gsm cardstock. Over 2200 droplets were created using the six different fluids and their final dried stain diameter was measured. Droplet spread was plotted using the Scheller and Bousfield correlation and uses effective viscosity as a parameter for non-Newtonian fluids. Comparing the results to an expected whole human blood range validated the spread of the candidate FBS sol-gel material in passive drip bloodstain pattern simulation. These findings complement the practical application of the material as a safe substitute for demonstrating droplet spread under controlled conditions on hard paper surfaces.

The application of silicon sol-gel technology to forensic blood substitute development: Mimicking aspects of whole human blood rheology.

Solution-gelation chemistry has promising applications in forensic synthetic blood substitute development. This research offers a silicon-based sol-gel approach to creating stable materials that share similar rheological properties to that of whole human blood samples. Room temperature, high water content, silicon sol-gels were created using the organosilane precursors 3-glycidoxypropyltrimethoxysilane and tetraethylorthosilicate along with various concentrations of filler and pigment. Shear-thinning non-Newtonian properties were observed within most formulations of the presented materials. The effects of colloidal concentration, temperature, age and filler addition on the viscosity of the sol-gels were investigated. SEM-EDS analysis was used to identify the behavior of the fillers within the film and support their inclusion for basic bloodstain pattern simulation. A final proposed candidate sol-gel was assessed using a previously reported passive drip simulation test on a hard, dry surface and passed. This works represents encouraging development in providing safe material alternatives to using whole human blood for forensic training and research.

Passive Drip Stain Formation Dynamics of Blood onto Hard Surfaces and Comparison with Simple Fluids for Blood Substitute Development and Assessment.

The spreading dynamics of blood dripping onto hard surfaces is compared to two spreading models. Samples of human blood, porcine blood, and Millipore® water were dripped onto cardboard, foamcore, and glass surfaces in low velocity passive drip simulations. Final stain diameter, the total number of spines and scallops, and angle of impact were measured and analyzed. Spreading is best predicted by applying the concept of effective viscosity to the Scheller and Bousfield (R2  = 0.91) and Roisman (R2  = 0.89) spreading models. In the tested conditions, blood spreads with Newtonian tendencies; however, has quantifiable differences in stain appearance to Newtonian fluids like water. This is encouraging for the development of water-based fluids as synthetic blood substitutes (SBSs). The work presents an assessment platform to quantify and score the performance of simple water-based fluids using final stain diameter (6 points) and number of spines and scallops (6 points) at six dripping heights between 20 and 120 cm. The angle of impact of a stain alone is not a sensitive measure of SBS performance, but stain formation scores the SBS's performance with another 1 point. Together these features generate a quantitative relative ranking system, of a maximum possible 13 points, that can be used to support the use of a particular fluid for the creation of a drip stain. The performance of twenty simple fluids in the simulated dripping assessment test is described.

An Impact Velocity Device Design for Blood Spatter Pattern Generation with Considerations for High-Speed Video Analysis.

A mechanical device that uses gravitational and spring compression forces to create spatter patterns of known impact velocities is presented and discussed. The custom-made device uses either two or four springs (k1 = 267.8 N/m, k2 = 535.5 N/m) in parallel to create seventeen reproducible impact velocities between 2.1 and 4.0 m/s. The impactor is held at several known spring extensions using an electromagnet. Trigger inputs to the high-speed video camera allow the user to control the magnet's release while capturing video footage simultaneously. A polycarbonate base is used to allow for simultaneous monitoring of the side and bottom views of the impact event. Twenty-four patterns were created across the impact velocity range and analyzed using HemoSpat. Area of origin estimations fell within an acceptable range (ΔXav = -5.5 ± 1.9 cm, ΔYav = -2.6 ± 2.8 cm, ΔZav = +5.5 ± 3.8 cm), supporting distribution analysis for the use in research or bloodstain pattern training. This work provides a framework for those interested in developing a robust impact device.

Three physical factors that affect the crown growth of the impact mechanism and its implications for bloodstain pattern analysis.

This research uses high-speed video analysis of bloodstain impact events to investigate the influence of impact velocity, fluid depth and free-space on the characteristics of the mechanism. We focus on the changes in the crown growth over time. This work demonstrates qualitative differences in the impact mechanism under a range of impact conditions. These differences are further explained quantitatively as a function of measured crown width and height lengths over time. Fluid dynamic explanations of this growth are featured in the results and discussion. A comparison to water dynamics is reported. Our image analysis demonstrates that droplets are consistently formed at points which are different from the impactor/fluid interface and that this difference is fluid dependent. This fluid dependency demonstrates the importance of accurately modeling fluid dynamics of blood when designing and deploying blood substitutes in forensics applications.

Novel silica sol-gel passive sampler for mercury monitoring in aqueous systems.

A novel passive sampler for mercury monitoring was prepared using organosilica sol-gel materials. It comprises a binding layer with thiol groups for mercury complexation and a porous diffusive layer through which mercury can diffuse and arrive at the binding layer. Our study demonstrated that this new sampler follows the principle of passive sampling. The mass of mercury accumulated in the binding layer depends linearly on the mercury concentration in solution, the sampling rate and the exposure time. A typical sol-gel sampler is characterized by a diffusive layer of 1.2 µm, in which mercury ions diffuse with a coefficient of D=0.09×10(-6) cm(2) s(-1), resulting in an uptake R(s) of 8.8 mL h(-1). The capacity for mercury uptake is approximately 0.64 µg cm(-2). Mercury diffusion and binding in the passive sampler are independent of the type of mercury-chloride complex, which potentially opens the door to use this device for mercury monitoring in a wide range of natural waters.