A preliminary study on the treatment of human blood soiled stubs, in presence of GSR particles.

In the investigation of gunshot deaths, Bloodstains Pattern Analysis (BPA) and, in particular, backspatter patterns found on the body of the suspect/victim and on the surfaces close to the entrance wound of the bullet can provide investigators with important indications on the dynamics of the events. Backspatter patterns have, however, morphological characteristics common to other bloodstains of different origin, so, in order to positively identify them, a possible solution is represented by their sampling, using an aluminum stub for electron microscopy, for the detection of gunshot residues (GSR) present. The latter, however, if present below the small blood crusts, could be difficult to detect during analysis by Scanning Electron Microscopy/Energy Dispersive X-ray microanalysis (SEM/EDX). In this preliminary study we propose the treatment of the stub surface with a solution based on sodium hypochlorite and calcium chloride, in order to remove/reduce the blood crusts present on the stub surface.

Nothing but hot air?-On the molecular ballistic analysis of backspatter generated by and the hazard potential of blank guns.

Blank cartridge guns are prevalent especially in countries with laws restricting access to conventional firearms, and it is a common misconception that these weapons are harmless and only used as toys or for intimidation. However, although their harming potential is well-documented by numerous reports of accidents, suicides, and homicides, a systematic molecular biological investigation of traces generated by shots from blank cartridges at biological targets has not been done so far. Herein, we investigate the occurrence and analyzability of backspatter generated by shots of different types of blank cartridge guns firing different types of blank ammunition at ballistic gelatin model cubes doped with human blood and radiological contrast agent soaked into a spongious matrix and covered with three different variants of skin simulants. All skin simulants were penetrated, and backspatter was created in 100% of the shots in amounts sufficient for forensic short tandem repeat (STR) typing that resulted in the correct identification of the respective blood donor. Visible backspatter was documented on the muzzle and/or inside the barrel in all cases, and in 75% of cases also on the outer surfaces and on the shooter's hand(s). Wound cavities were measured and ranged between 1 and 4.5 cm in depth. Discussing our findings, we provide recommendations for finding, recovering, and analyzing trace material from blank guns, and we demonstrate the considerable hazard potential of these devices, which is further emphasized by the presentation of a comprehensive overview of the pertinent literature on injuries inflicted by blank guns.

Ten years of molecular ballistics-a review and a field guide.

Molecular ballistics combines molecular biological, forensic ballistic, and wound ballistic insights and approaches in the description, collection, objective investigation, and contextualization of the complex patterns of biological evidence that are generated by gunshots at biological targets. Setting out in 2010 with two seminal publications proving the principle that DNA from backspatter collected from inside surfaces of firearms can be retreived and successfully be analyzed, molecular ballistics covered a lot of ground until today. In this review, 10 years later, we begin with a comprehensive description and brief history of the field and lay out its intersections with other forensic disciplines like wound ballistics, forensic molecular biology, blood pattern analysis, and crime scene investigation. In an application guide section, we aim to raise consciousness to backspatter traces and the inside surfaces of firearms as sources of forensic evidence. Covering crime scene practical as well as forensic genetic aspects, we introduce operational requirements and lay out possible procedures, including forensic RNA analysis, when searching for, collecting, analyzing, and contextualizing such trace material. We discuss the intricacies and rationales of ballistic model building, employing different tissue, skin, and bone simulants and the advantages of the "triple-contrast" method in molecular ballistics and give advice on how to stage experimental shootings in molecular ballistic research. Finally, we take a look at future applications and prospects of molecular ballistics.

Study of backspatter using high-speed video of experimental gunshots.

Backspatter is biological material that is ejected from the entry wound against the line of fire. This phenomenon was also observed in wound ballistic simulations using so called "reference cubes" (12 cm edge length, 10% gelatin, 4 °C, paint pad beneath the cover). High-speed video records from 102 experimental shots to these target models using full metal jacketed bullets in the calibers .32 auto, .38 special, 9 mm Luger and .357 Magnum were analyzed for chronology, morphological appearance and velocity of fluid ejection. Generally, a short tail splashing of surface material occurred when the bullet was penetrating the target. In 51 shots from distance (≥ 5 cm), regardless of caliber and shot range, a linear jet of fluid started in connection with the first collapse of the temporary cavity. The initial velocity of the jet was measured between 6 and 45 m/s. The jet was streaming on for about 60 to 100 ms with a stochastic deviation of ± 13° to the horizontal. Close range and contact shots showed earlier and faster (up to 330 m/s) backspatter depending on the cartridge and the gap between muzzle and target. Gaseous aerosol-like spray and cone-like spatter indicated an increasing influence of muzzle gases with decreasing shot range. Even under standardized experimental conditions, variations of backspatter were observed in near/contact shots.

DNA-free does not mean RNA-free-The unwanted persistence of RNA.

Contact shots to the head often provoke a transfer of biological traces into firearm barrels, which are not visible at endoscopic inspection. STR-PCR can amplify these latent traces and assign them to the victim. Via RNA-DNA-co-extraction also miRNA can be detected, which allow a conclusion to be drawn about the body fluid or tissue. Molecular genetic analysis of experimental stains in firearm barrels requires the guarantee that the barrel is initially free of any nucleic acid. Twelve shots were fired to so-called "reference cubes" (10 % gelatine, 12 cm edge length, embedded paint-blood-pad) using three current handguns: from 20 and 30 cm distance, four at close range (1-2.5 cm) and six contact shots. After endoscopic examination and swabbing of the barrels, a previously described mechanical and chemical cleaning using DNAExitusPlus™ was performed. The inner surface of the barrel was thoroughly wiped off using moistened forensic swabs, which were submitted to RNA-DNA-co-extraction. The combined thorough mechanical cleaning with Ballistol® and the application of DNAExitusPlus™ eliminated any profilable DNA in all samples. However, in 10 of 12 samples RNA concentrations between 0.11 - 0.79 ng/µl were measured. Furthermore, in 9 of 12 samples blood-specific miRNA (miR-451a) was detected. Summarizing, none of the experimentally contaminated barrels was RNA-free despite the performed cleaning procedure. Further investigation showed, that even "professional" cleaning by a gunsmith did not remove RNA.

A distant relationship?-investigation of correlations between DNA isolated from backspatter traces recovered from firearms, wound profile characteristics, and shooting distance.

In molecular ballistics, where traces originating from the use of firearms against biological targets are investigated, "backspatter" traces are of particular importance. This biological material comprising blood and tissue from the victim is propelled back from the bullet entry site towards the direction of the shooter and can consolidate and persist on the inner and outer surfaces of the firearm, from where it can be collected and analyzed. Thus, a connection between the weapon and the victim can be established solely by molecular biological trace analysis. For the criminalistic investigation of gun-related crimes, the determination of the distance between the weapon and the victim can be of critical importance in reconstructing the circumstances of a crime. In this study, we investigated possible correlations between the shooting distance and the amount of backspatter in/on the used firearm. To this purpose, we employed a previously established skull model and performed shootings in triplicates from various distances up to 50 cm with two types of handguns (pistol and revolver). Backspatter was collected from various sampling locations, and DNA contents were quantified. A post-shooting wound channel evaluation was conducted by optical and radiological evaluation. The obtained DNA yields varied considerably between replicates from the same and from different distances. In contrast, apart from contact shots, no meaningful differences were observable in wound channel evaluations. In summary, no meaningful correlation between backspatter distribution and DNA yields, the shooting distance and the condition of the wound channel could be established.

The influence of muzzle gas on the temporary cavity.

Shot range, the muzzle-target distance, is a crucial parameter for forensic reconstruction of deaths by firearms. In a large number of cases, especially suicides, the forensic pathologist is confronted with contact or near-contact shots, where muzzle gases play an additional role. This study was conducted to systematically investigate the influence of muzzle gases on the temporary cavity (TC). A total of 72 shots were fired using full metal-jacketed bullets in four forensically relevant calibres from 10-, 5-, 3-, 2- and 1-cm distance and in close contact. Target model was the so-called reference cube (10% gelatine at 4 °C) with 12-cm edge length. The TC was recorded using high-speed video (HSV). Cross-sectional analysis was performed by cutting the blocks to 1-cm slices, which were evaluated by applying the polygon method. The TC of shots from 10 and 5 cm distance had a tubular form. This aspect changed depending on the cartridge with decreasing distance (≤ 3 cm) into a pear-like form, which was typical for contact shots. The cumulated heights of the TC increased with decreasing distance below 3 cm. Contact shots approximately doubled the extension of the TC compared with exclusive energy transfer. Whereas HSV documented an increasingly asymmetric profile with ballooning at the entry side, cross-sectional analysis of cracks in gelatine resulted in convex graphs with only slight asymmetry for contact shots. Additional damage in gelatine was detected for 3-cm distance or less in calibre .357 Magnum and ≤ 2 cm for .32 auto, .38 special and 9mm Luger. The increasing influence of muzzle gas pressure is detectable with decreasing shot range below 3 cm.

Evaluation of the backspatter generation and wound profiles of an anatomically correct skull model for molecular ballistics.

Molecular ballistics connects the molecular genetic analysis of biological traces with the wounding events and complex forensic traces investigated in terminal ballistics. Backspatter, which originates from a projectile hitting a biological target when blood and/or tissue is propelled back into the direction of the gun, is of particular interest; those traces can consolidate and persist on the outer and inner surfaces of firearms and serve as evidence in criminal investigations. Herein, we are the first to present an anatomically correct head model for molecular ballistic research based on a polyurethane skull replica enclosing tissue-simulating sponge material that is doped with "triple-contrast" mixture (EDTA-blood, acrylic paint, and an x-ray contrast agent). Ten percent ballistic gelatin was used as brain simulant. We conducted contact and intermediate-range shots with a Glock 19 pistol (9 mm Luger), a pump-action shotgun (12/70 slugs), and blank cartridge handguns. Each shot was documented by a high-speed camera at 35,000 fps. Apart from the blank cartridge guns, all gunshots penetrated the skull model and created backspatter, which was recovered from the distal part of the barrels and analyzed. The pistol contact shots and one of three shotgun shots yielded full STR profiles. While the shotgun slugs destroyed the skulls, the remaining models could be used for radiological and optical fracture and wound channel evaluation. Known backspatter mechanisms and their respective timing could be confirmed visually by video analysis. Our complete model setup proved to be well applicable to molecular ballistic research as well as wound channel and fracture pattern investigation.

Distortion of the temporary cavity and its influence on staining in firearm barrels.

After contact shots to the head, biological traces can be found inside the barrel of the firearm. Experimental protocols to generate this sort of staining, using 12 cm gelatin cubes containing thin foil bags filled with acrylic paint, human blood, and radiocontrast agent, have been developed. Previous research on shots fired at a distance has shown the underlay sustaining these gelatin cubes has an influence on experimental results. This study was conducted to investigate the role of the sustaining base of the gelatin blocks during contact shots, and its influence on the staining result inside firearm barrels. Eighteen contact shots were performed using 22 LR, 32 ACP (7.65 Browning) and 9 mm Luger semi-automatic pistols. With each pistol, shots were fired onto six gelatin cubes; three placed upon a rigid platform and three upon an elastic underlay. The shots were recorded by a high-speed video camera as they penetrated the gelatin cube. Any staining present inside the firearm barrels after the shots were fired was documented by endoscopy. Cross sections of the gelatin blocks were then compared to the high-speed video. It was found that the nature of the staining inside the barrel was not influenced by the underlay sustaining the target model. In the experiment using a 9 mm Luger, the rigid counterfort provoked a visible distortion of the temporary cavity, but, cross sectional analysis of the gelatin cubes did not reveal a relevant influence of the sustaining underlay on the crack length in the gelatin. This could be explained by a secondary expansion of the temporary cavity left by the projectile as a consequence of subsequent inflow of muzzle gases.

Cranial Backspatter Pattern Production Utilizing Human Cadavers.

A backspatter pattern results from blood drops that travel retrograde to an applied external force. Historically, an array of animals and nonhuman objects have been used to create and study backspatter patterns. In this study, backspatter patterns captured on foam core targets that were placed 45.72 cm (18 in) behind the impact site (occipital area of the skull) were produced by cranial gunshots to human cadavers that were reinfused with fresh defibrinated bovine blood. These patterns were compared to the backspatter patterns produced by shooting blood-soaked sponges, a typical simulant used in controlled studies of backspatter pattern production and characteristics. The backspatter pattern produced by shooting an actual human head was found to be different than those of blood-soaked sponges in the number of stains produced, the size and size range of the stains, and the stain dispersion patterns.

RNA/DNA co-analysis on aged bloodstains from adhesive tapes used for gunshot residue collection from hands.

In cases of firearm related fatalities a systematic investigation at the scene of death is indispensable to differentiate between self-inflicted and homicidal gunshot injuries. A common method to preserve gunshot residues (GSR) is their collection using adhesive tapes. However, the biological material gathered at the same time by the tapes would be of special interest if backspatter, ejected from the entrance wound against the direction of fire, could be detected. In the present study we examined the success rate of co-analysis of RNA and DNA recovered from biological traces sampled with adhesive tapes. The material originated from eight cases of fatal gunshots, taken from the hands of suspects or victims, examined 5 to 19 years ago for GSR. For all types of adhesive tapes tested, quantity and quality of the co-extracted nucleic acids was insufficient for successful DNA profiling, but was sufficient for the detection of blood-specific micro RNA (miRNA). In summary, sampling trace evidence from the hands of persons involved in fatal gunshots with adhesive tapes has a long-term detrimental effect on biological traces.

The reference cube: A new ballistic model to generate staining in firearm barrels.

After contact shots to the head biological traces can be found inside firearm barrels. So far silicone coated, gelatin filled box models were used to generate such staining according to the triple contrast method (mixture of acrylic paint, barium sulfate and blood sealed in a thin foil bag). This study was conducted to develop a transparent ballistic model allowing contact shots. Gelatin filled polyethylene bottles with and without a silicone coat were tested in comparison to non-covered gelatin blocks. Finally, thin foil bags of 5 cm × 5 cm dimension were glued on a synthetic absorbent kitchen wipe on top of which 1 L 10% gelatin solution was molded to create blocks of 8.5 cm length. A kitchen wipe with a paint pad on its inside formed the front of the cube. Three contact shots each with a 9 mm Luger pistol and a .38 special revolver were performed on all model variations. The staining was documented by endoscopy and swabs gathered from both ends of the barrel were analyzed by quantitative PCR. Reliable staining was achieved using the front covered gelatin block with comparable results to the silicone coated box model used before. For further research using ballistic models to simulate a human head a symmetric form of the gelatin block such as a cube is recommended.

Staining in firearm barrels after experimental contact shots.

After contact shots to the head biological traces inside firearm barrels can be found. This study was conducted to simulate and to evaluate such staining. Five current handguns of four inch barrel length in the calibre .22 long rifle, 7.65mm Browning, 9mm Luger and .38 special were used to perform 24 contact shots on silicone coated, gelatine filled box models using the triple contrast method. The staining was documented by endoscopy and swabs gathered from both ends of the barrel were analysed by quantitative PCR. With the exception of the .22 revolver, all firearms showed distinct staining which decreased from the muzzle to the rear end of the barrel. The pattern was varied, showing droplets, elongated forms or stripes. In 14 of 24 shots, staining reached the chamber. The staining results were comparable to real suicide cases.

Visualization of the powder pocket and its influence on staining in firearm barrels in experimental contact shots.

The powder pocket or soot cavity is a morphologic characteristic of a close contact shot. In a research project concerning staining inside the barrel, the influence of the powder pocket on these traces was investigated.According to the 'triple contrast method', thin pads containing a mixture of acrylic paint, radiocontrast agent and blood were glued on plastic boxes which were coated with a 3-4-mm-thick silicone layer. The containers filled with 10 % ballistic gelatine, were stored for at least 60 h at 4 °C. Thirty-three contact shots were realized using different pistols and 22 lr, .32 auto, .38 special and 9-mm Luger with different barrel length using subsonic, non-deforming ammunition.The documentation comprised endoscopy, high speed video and computer tomography (CT) of the target models. Using image analysis, the ballooning of the silicone coat was studied (lateral view projection).High-speed video confirmed the actual comprehension of the behaviour of muzzle gases in contact shots. The powder cavity rises in about 1.5 to 2 ms, preceding the maximum of the temporary cavity, and the powder pocket's collapse takes 2.5 to 3 ms.The size of the silicone dome increased with decreasing barrel length. Comparing semi-automatic pistols of 4 in. barrel length in the calibres, .32 auto and 9-mm Luger, there were no significant differences of the powder pocket size. Material transport was observed, against and perpendicularly to the shooting direction. CT showed undermining and gas inclusions inside the powder pocket. A correlation between amount and pattern of the staining inside the barrel and the volume of the powder pocket was not observed.

Simulating backspatter of blood from cranial gunshot wounds using pig models.

Few studies have examined the biomechanical basis for backspatter from cranial gunshot wounds. Backspatter is material which travels against the direction of fire following ejection from a gunshot entrance wound. Our paper focuses on the use of animals for reconstructing this phenomenon. Five live pigs and several slaughtered pigs were shot using either 9 × 19 mm, 115 grain, full metal jacketed ammunition or .22 long rifle, 40 grain, lead, round-nose ammunition. A high-speed camera was used to record the entrance wound formation and backspatter. A small amount of backspattered material was produced with all targets, and blood backspatter was seen in a few cases. However, we conclude that our model provides an understanding of the phenomenon of backspatter and the physical mechanisms associated with it. The various components of the mechanism of backspatter formation are complex and overlap. The principle mechanism observed in pig cranial gunshots was the high-speed impact response of the skin overlying the skull bone. This study has also produced evidence supporting the view that backspatter can result from the splashing of superficial blood if it is already present on the skin. Subcutaneous gas effects have been demonstrated for backspatter from contact shots. There has been no clear evidence of the role of the collapse of a temporary cavity within the brain.

How far does it get?--The effect of shooting distance and type of firearm on the simultaneous analysis of DNA and RNA from backspatter recovered from inside and outside surfaces of firearms.

When a firearm projectile hits a biological target a spray of biological material (e.g. blood and tissue) is ejected from the entrance wound and propelled back into the direction of the firearm. This phenomenon has been termed 'backspatter' and if backspattered biological material reaches the firearm on its backward trajectory it may persist on and be recovered from the firearm's inside surfaces. Molecular genetic analysis of backspatter generated by contact shots and shots from very short distances has already been demonstrated to critically contribute to victim identification and the reconstruction of firearm-related crimes. It is not known, however, up to what shooting distance can backspatter be found on firearms' inside surfaces and what influence the weapon's type and caliber has on backspatter attributes (e.g. reach, amount and distribution). Therefore, the present pilot study investigated the effect of serval combinations of shooting distances and types of firearms and ammunitions on the analyzability of co-extracted DNA and micro-RNA in samples of backspatter collected from interior and exterior surfaces of the firearms after experimental shootings employing standardized ballistic models. We demonstrate the limiting effect of shooting distance and the type of firearm on the yield of nucleic acids recovered from backspatter and the success rates of forensic DNA profiling and RNA based body-fluid and organ tissue identification in experimental shootings.

Evaluating simulant materials for understanding cranial backspatter from a ballistic projectile.

In cranial wounds resulting from a gunshot, the study of backspatter patterns can provide information about the actual incidents by linking material to surrounding objects. This study investigates the physics of backspatter from a high-speed projectile impact and evaluates a range of simulant materials using impact tests. Next, we evaluate a mesh-free method called smoothed particle hydrodynamics (SPH) to model the splashing mechanism during backspatter. The study has shown that a projectile impact causes fragmentation at the impact site, while transferring momentum to fragmented particles. The particles travel along the path of least resistance, leading to partial material movement in the reverse direction of the projectile motion causing backspatter. Medium-density fiberboard is a better simulant for a human skull than polycarbonate, and lorica leather is a better simulant for a human skin than natural rubber. SPH is an effective numerical method for modeling the high-speed impact fracture and fragmentations.

Characteristics of backspatter on the firearm and shooting hand--an experimental analysis of close-range gunshots.

Contact and close-range gunshot injuries can produce bloodstains on the shooting hand and weapon. In this study, backspatter was analyzed in experimental horizontal pistol shots into blood-filled sponges. The shooting distances were set between 0 and 45 cm. With increasing shooting distance, there was a significant decrease in the degree of backspatter. Microspatter could be detected on the weapon and shooting hand up to a shooting distance of 40 cm. They were predominantly located on thumb and index finger as well as the radial dorsal aspects of the hand. Fine spray of microspatter combined with elongated droplets in the shape of exclamation marks was found at contact and 2-cm distance shots. An angular calculation based on the shape of the bloodstains was possible up to a shooting distance of 2 cm. No bloodstains were detected beyond 45 cm.

Visualization of the air ejected from the temporary cavity in brain and tissue simulants during gunshot wounding.

One hypothesis for the physical mechanism responsible for backspatter during cranial gunshot wounding is that air is ejected by the collapse of the temporary cavity formed around the bullet path. Using bovine and ovine heads and simulant materials, evidence of this ejection was sought by measuring the velocity of the air that was drawn in and ejected from the cavity in front of the wound channel after bullet impact. A laminar flow of fog-laden air was arranged in front of the wound channel and two high speed cameras recording at 30,000 frames/second captured the air motion. All samples were shot with standard 9 mm × 19 mm FMJ ammunition. Different concentrations of ballistic gelatine were used to characterize the effect of elasticity of the material on the velocity of the air. Fresh bovine and ovine heads were shot with the same experimental set up to investigate if there was induction of air into, and ejection of air from the entrance wounds. The results show, for the first time, that the temporary cavity does eject air in gelatine. The velocity of in-drawn air for 3, 5 and 10% concentration of gelatine was 81, 76 and 65 m/s respectively and the velocity of ejected air for 5 and 10% concentration of gelatine were 43 and 72 m/s respectively. The results show that when the concentration of gelatine is increased, the velocity of the air drawn into the cavity decreases and the velocity of the ejected air increases. However, no ejection was observed in 3% gelatine, ovine or bovine heads. Although ejection of air was not observed, ejection of brain from the wound channel was seen. Using the velocity of the ejected brain, the minimum intracranial pressure required to eject the brain tissue was estimated to be 712 kPa and 468 kPa for the sheep and bovine heads respectively.

Experimental investigation of the mechanical properties of brain simulants used for cranial gunshot simulation.

The mechanical properties of the human brain at high strain rate were investigated to analyse the mechanisms that cause backspatter when a cranial gunshot wound occurs. Different concentrations of gelatine and a new material (M1) developed in this work were tested and compared to bovine brain samples. Kinetic energy absorption and expansion rate of the samples caused by the impact of a bullet from .22 air rifle (AR) (average velocity (uav) of 290m/s) and .22 long rifle (LR) (average velocity (uav) of 330m/s) were analysed using a high speed camera (24,000fps). The AR projectile had, in the region of interest, an average kinetic energy (Ek) of 42±1.3J. On average, the bovine brain absorbed 50±5% of Ek, and the simulants 46-58±5%. The Ek of the .22 LR was 141±3.7J. The bovine brain absorbed 27% of the .22LR Ek and the simulants 15-29%. The expansion of the sample, after penetration, was measured. The bovine brain experienced significant plastic deformation whereas the gelatine solution exhibited a principally elastic response. The permanent damage patterns in the M1 material were much closer to those in brain tissue, than were the damage patterns in the gelatine. The results provide a first step to developing a realistic experimental simulant for the human brain which can produce the same blood backspatter patterns as a human brain during a cranial gunshot. These results can also be used to improve the 3D models of human heads used in car crash and blast trauma injury research.