Changes in the morphology and presumptive chemistry of impact and pooled bloodstain patterns by Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Bloodstain pattern analysis can be critical to accurate crime scene reconstruction. However, bloodstain patterns can be altered in the presence of insects and can confound crime scene reconstruction. To address this problem, we conducted a series of controlled laboratory experiments to investigate the effect of Lucilia sericata (Meigen) on impact bloodstains and pooled bloodstains in association with three combinations of common surfaces (linoleum/painted drywall, wood floor/wallpaper, and carpet/wood paneling). L. sericata fed from the pooled bloodstains and added insect stains through regurgitation and defecation of consumed blood. L. sericata formed defecatory trails of insect stains that indicated directionality. Defecatory stains fluoresced when viewed at 465 nm with an orange filter. These observations differed from Calliphora vicina insect stains because feeding on blood spatter was not observed and trails of insect stains were formed by L. sericata. The fluorescence of defecatory stains can be used as a method to detect insect stains and discriminate them from real bloodstains.     

Volume Determination of Fresh and Dried Bloodstains by Means of Optical Coherence Tomography

The volume of bloodstains found on crime scenes may help forensic investigators reconstruct the location and kinematics of bloodletting events, as stain size, volume, and impact velocity are related. Optical coherence tomography was used as a method to determine the volume and volume ratio of dried and fresh bloodstains on both glass and irregular surfaces or deposited with an impact velocity. The volume of blood drops deposited on smooth glass surfaces was measured within a deviation of 2%. This deviation increased for droplets on irregular surfaces or deposited with an impact velocity. The volume ratio of dried and fresh bloodstains was equal to 19–28% depending on the individual donor and on the use of an anticoagulant. Optical coherence tomography is a good method to determine the volume of fresh and dried bloodstains in laboratory conditions and allows accurate determination of the dry/fresh ratio.     

The Morphology of Fecal and Regurgitation Artifacts Deposited by the Blow Fly Lucilia cuprina Fed a Diet of Human Blood

Fly feces and regurgitation deposits may be mistaken for bloodstain patterns at a crime scene, potentially compromising event reconstruction and/or misdirecting police resources. In some instances, these artifacts contain sufficient human biological material to generate a full DNA profile, sometimes 2 years after deposition. Clearly, it is important that investigators can make the distinction between artifacts and bloodstains. This study examined 6645 artifacts deposited on a smooth, nonporous surface after Lucilia cuprina were fed human blood. Artifacts were also compared with bloodstains on a variety of other surfaces. Both similarities and differences were found between artifacts and bloodstains, highlighting the need for an identification system to assist personnel with little training in bloodstain pattern analysis. The morphology of the artifacts has been described so that these deposits may be more clearly distinguished from bloodstains, targeted by crime scene personnel as potential sources of human DNA, and/or identified as potential evidence contaminants. Flowcharts have been devised to facilitate the analysis.     

Validation studies of rapid stain identification-blood (RSID-blood) kit in forensic caseworks

When bloodstains are detected at crime scene using presumptive tests (e.g. luminol, phenolphthalein, leuchomalachite green), it is important to establish the real human nature of each stain. This is possible using confirmatory tests. One of these is rapid stain identification-blood (RISD-blood) a lateral flow immuno-chromatographic strip test format which allows the identification of human blood by detection of glycophorin A, a red blood cell membrane antigen, using two anti-human glycophorin A (GPA) monoclonal antibodies.The aim of this study is to assess the sensitivity of RSID-blood test in old, degraded bloodstains and in some bloodstains previously treated with BlueStar Forensic, a presumptive test which is often used in crime scene investigations to detect latent bloodstains. The genetic analysis of all bloodstains of confirmed human nature was subsequently performed using the AmpF1STR Identifiler PCR Amplification Kit (Applied Biosystems), to validate the possibility of obtain a consistent and reliable DNA typing results.     

The Accuracy and Repeatability of Reconstructing Single Bullet Impacts Using the 2D Ellipse Method

When a bullet punctures a surface, it leaves behind a bullet impact, which can be analyzed in order to determine the origin and trajectory path from which a bullet was discharged using many different scientific methods to reconstruct a shooting scene. The purpose of the research was to test the accuracy and repeatability of reconstructing the impact angle of single bullet impacts using the ellipse method. The research was conducted by firing various calibers of ammunition into drywall panels positioned at varying angles of incidence from 88° until ricochet occurred. This was done in order to determine which caliber type and angle of incidence are most accurate and precise for reconstructing a shooting scene. The study examined four caliber types fired into drywall panels, with 5 repeated shots for each of the 11 angles of incidence (n = 220). Furthermore, 31 participants partook in the study to estimate a bullet impact angle of incidence utilizing the ellipse method. The results show that the best performance (accuracy and repeatability) is seen with the measurements of the 0.45 caliber ammunition. When angle of incidences is low (<64°), the performance (accuracy and repeatability) was seen to be better in all caliber ammunitions. Overall, the data provided for single bullet impacts deposited in drywall show that the ellipse method is useful in providing measurements for most crime scene reconstruction purposes and has also demonstrated that results vary depending on the type of ammunition, firearm, and angle of incidence being examined.     

STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes

Improvements in detection limits/sensitivity and lower sample consumption are potential benefits of reducing PCR reaction volumes used in forensic DNA typing of crime scene samples. This premise was studied first with experimental mixtures and a nine-loci megaplex, which demonstrated stochiometric amplification and accurate detection. Next, adjudicated casework samples were subjected to amplification under 15 different template DNA to PCR reaction volume ratios. Reduction of PCR reaction volume and DNA down to 10 microL and 0.500 ng, respectively, produced identical profiles with the same signal intensity and heterozygous allele peak height ratio (HR). Reduction to 5 microL and 0.063 ng yielded HR values that were slightly affected in one to three STR loci. PCR reaction volume reduction can enhance detection and sensitivity while reducing the consumption of irreplaceable crime scene samples.     

Morphology of low-velocity impact stains produced from single drops of blood

Systematic variation of blood droplet volume, the distance fallen and the surface (paper, wood, plastics, tiles) led to the conclusion that the size and the shape of the stains ("fingers", satellites) allowed to deduce the distance fallen but only if the actual surface structure was known. We found that detailed photography at the crime scene was necessary, yet experiments have to be performed due to the extreme influence of the actual surface texture on all characteristics (size, spines, peripheral spatter) of the blood stains.     

Deducing drop size and impact velocity from circular bloodstains

An experimental study was done to determine the diameter and velocity of blood drops falling on a surface by measuring the size of bloodstains they produced and counting the number of radial spines projecting from them. Bloodstains were formed by releasing drops of pig blood with a range of diameters (3.0-4.3 mm) and impact velocities (2.4-4.9 m/s), onto four different flat surfaces (glass, steel, plastic, paper) with varying roughness (0.03-2.9 microm). High-speed photography was used to record drop impact dynamics. Bloodstain diameters and the number of spines formed around the rim of stains increased with impact velocity and drop diameter. Increasing surface roughness reduced stain diameter and promoted merging of spines, diminishing their number. Equations are presented that explicitly relate drop diameter and impact velocity to measurements of stain diameter and number of spines.     

圆形滴状血迹形态与血滴体积、撞击速度的相关性

目的观察不同体积的血滴以3种撞击速度垂直撞击3种不同介质形成的滴状血迹形态及大小,并探讨其相关性。方法将8种不同体积（14～115IxL）的抗凝猪m形成血滴,以3种不同的撞击速度（3．1～5．4m／s）分别垂直滴落于白纸、玻璃及釉面地砖上,观察形成血迹的形态特征,测量其直径,并采用SPSS13．0统计软件进行分析。结果在3种撞击速度条件下,在3种介质上形成的滴状血迹直径均随血滴体积的增加而增大,血迹直径与血滴体积存在较好的线性关系,可得出回归方程（回归模型R2〉0．85,P〈0．01）;介质表面越粗糙,血迹直径越小;仅在自纸上发现血迹边缘有突起形成,且其数目随血滴体积的增加而增多。撞击速度及血滴体积存在较好的多重线性关系,可得出多重回归方程（回归模型R2〉0．90,P〈0．01）。结论垂直撞击形成的血迹形态及大小与血滴体积、撞击速度具有显著的相关性,可望用于推测出血源位置、受伤体位及出血部位载体。     

Alteration of expirated bloodstain patterns by Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae) through ingestion and deposition of artifacts

Abstract: Bloodstain pattern analysis can provide insight into a sequence of events associated with a violent crime. However, bloodstain pattern analysis can be confounded by the feeding activity of blow flies. We conducted two laboratory experiments to investigate the relationships between Lucilia sericata (green bottle fly) and Calliphora vicina (blue bottle fly), expirated bloodstains, and pooled bloodstains on a range of surfaces (linoleum, wallpaper, textured paint). C. vicina and L. sericata changed bloodstain pattern morphology through feeding and defecation. They also deposited artifacts in rooms where blood was not present originally. Chemical presumptive tests (Hemastix^®, phenolphthalein, leucocrystal violet, fluorescein) were not able to differentiate between insect artifacts and bloodstains. Thus, C. vicina and L. sericata can confound bloodstain pattern analysis, crime scene investigation, and reconstruction. Crime scene investigators should be aware of these fundamental behaviors, and the effects that blow flies can have on expirated and pooled bloodstain patterns.     

“Bubbles”—A Spot Diagnosis

Abstract: Aspiration of blood is a phenomenon observed in violent and natural death scenarios. Bloodstain patterns evolving from expectoration of aspired blood may look suspicious of a violent genesis and thus mislead crime scene investigators. In the present case, a woman was found lying in a pool of blood on the kitchen floor. Furthermore, bloodstains covered her face, clothing, and surrounding furniture and walls. Bloodstain pattern analysis and medicolegal inspection of the suspected scene of crime were carried out and revealed dispersed stains with enclosed gas bubbles in the absence of signs of physical violence leading to the assessment of a natural manner of death. The bloodstains were attributed to expiration of blood because of an internal bleeding. Medicolegal autopsy confirmed the on‐site diagnosis as a fatal esophageal varix rupture was found.     

现场血迹推断出血点位置的研究进展

血迹是命案现场最常见的痕迹之一,对血迹形态的分析是一种重建犯罪现场的有效方法,而根据现场血迹推断出血点位置则是血迹形态分析的主要任务之一。随着血迹研究的不断发展,出现了许多不同的方法（如传统拉线法、三角函数法、绘图法、计算机程序法、公式计算法、回归方程法等）用于推断出血点位置。通过结合国内外相关文献,对不同的方法进行了综述,以期为相关研究提供参考。     

Powdered Activated Carbon: An Alternative Approach to Genomic DNA Purification

Forensic evidence samples are routinely found as stains on various substrates, which may contain substances known to inhibit polymerase chain reaction (PCR). The goal of this study was to evaluate post‐Chelex^®100 purification using powdered activated carbon (PAC). Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the DNA recovery and inhibitor removal using PAC with those of the Amicon^®Ultra 100K. For extracted bloodstains on soil and wood substrates, PAC and Amicon^®Ultra 100K generated similar DNA yield and quality. Moreover, the two methods significantly decreased the concentration of humic substances and tannins compared to nonpurified extracts (p < 0.001). In instances where extracts contained indigo dye (bloodstains on denim), Amicon^®Ultra 100K performed better than PAC due to improved amplifiability. Efficient adsorption of humic substances and tannins, which are common inhibitors, indicates PAC's potential application in the purification of high‐template DNA extracts.     

Impairment based legislative limits for driving under the influence of non-alcohol drugs in Norway

Bloodstains at crime scenes are among the most important types of evidence for forensic investigators. They can be used for DNA-profiling for verifying the suspect's identity or for pattern analysis in order to reconstruct the crime. However, until now, using bloodstains to determine the time elapsed since the crime was committed is still not possible. From a criminalistic point of view, an accurate estimation of when the crime was committed enables to verify witnesses' statements, limits the number of suspects and assesses alibis. Despite several attempts and exploration of many technologies during a century, no method has been materialized into forensic practice. This review gives an overview of an extensive search in scientific literature of techniques that address the quest for age determination of bloodstains. We found that most techniques are complementary to each other, in short as well as long term age determination. Techniques are compared concerning their sensitivity for short and long term ageing of bloodstains and concerning their possible applicability to be used on a crime scene. In addition, experimental challenges like substrate variation, interdonor variation and environmental influences are addressed. Comparison of these techniques contributes to our knowledge of the physics and biochemistry in an ageing bloodstain. Further improvement and incorporation of environmental factors are necessary to enable age determination of bloodstains to be acceptable in court.     

Blood droplet dynamics--I

The interpretation of bloodstain patterns at crime scenes has received increased attention in recent years. Important to an understanding of this is knowledge of the fundamentals of blood droplet formation and impact dynamics. A review of the literature reveals that a considerable amount of work has been done with aqueous drop dynamics. Workers in the forensic science area seem to have been unaware of this. In addition, some of the most important and comprehensive early work with blood droplet dynamics seems to have been forgotten. It is not cited in more recent publications dealing with bloodstain pattern interpretation. This literature is reviewed and discussed as well. The present study presents results of experiments with blood droplet dynamics and high-speed photographs of blood droplet impacts on stationary target surfaces. Some longstanding misconceptions of importance to forensic scientists engaged in crime scene reconstruction are discussed.     

Bloodstain pattern analysis--casework experience

The morphology of bloodstain distribution patterns at the crime scene carries vital information for a reconstruction of the events. Contrary to experimental work, case reports where the reconstruction has been verified have rarely been published. This is the reason why a series of four illustrative cases is presented where bloodstain pattern analysis at the crime scene made a reconstruction of the events possible and where this reconstruction was later verified by a confession of the offender. The cases include various types of bloodstains such as contact and smear stains, drop stains, arterial blood spatter and splash stains from both impact and cast-off pattern. Problems frequently encountered in practical casework are addressed, such as unfavourable environmental conditions or combinations of different bloodstain patterns. It is also demonstrated that the analysis of bloodstain morphology can support individualisation of stains by directing the selection of a limited number of stains from a complex pattern for DNA analysis. The complexity of real situations suggests a step-by-step approach starting with a comprehensive view of the overall picture. This is followed by a differentiation and analysis of single bloodstain patterns and a search for informative details. It is ideal when the expert inspecting the crime scene has also performed the autopsy, but he definitely must have detailed knowledge of the injuries of the deceased/injured and of the possible mechanisms of production.     

Development of a radioimmunoassay technique for the detection of human hemoglobin in dried bloodstains

A sensitive radioimmunoassay for the detection of human hemoglobin in dried bloodstains for the purpose of forensic science species identification has been developed. Bloodstains from 13 animal species were tested and found to be negative for human blood. A minimum volume of 0.8 microL of fresh blood is required to produce sufficient stain for successful testing. Bloodstains prepared from newborn and sickle-cell bloods were determined to be human. Bloodstains ranging in age from 1 month to 6 years which had been maintained desiccated at 20 to 25 degrees C were also successfully tested. Positive results were obtained on human bloodstains stored at 24 degrees C with relative humidity ranging from 0 to 98% for a period of 3 weeks. Absolute counts per minute (CPM) decreased with increased humidity. Human bloodstains exposed to bacterial contamination (gram positive or negative species) under humid conditions for 2 weeks also tested positive. Bacterial contamination caused a decrease in CPM, but insufficient to result in an erroneous conclusion as to species of origin. Positive results were also obtained on human bloodstains stored for 6 weeks at various temperatures ranging from -16 to 37 degrees C. No significant decreases in CPM were noted for any of the temperature conditions described.     

Fundamental studies of bloodstain formation and characteristics

A detailed understanding of blood droplet impact dynamics and stain formation is an essential prerequisite to the interpretation of both individual bloodstains and spatter patterns. The current literature on theoretical models for the spreading and splashing of liquid drops on surfaces relevant to the forensic context of bloodstain formation has been reviewed. These models have been evaluated for a paper substrate using experimental data obtained as function of droplet size, impact velocity and angle. It is shown that for perpendicular impact there are fairly simple mathematical models for the spreading diameter and the number of scallops or spines formed around the stain though these have quite limited ranges of validity in their basic form. In particular, predictions for the diameter are best for small droplets impacting at high velocity and the number of spines saturates for higher impact velocities. In the case of spreading, a modification to the energy conservation model is found to provide excellent agreement with experimental stain diameters across a wide range of impact velocities. For non-perpendicular impact, the width of stains is found to depend principally on the normal component of impact velocity and may be predicted by an appropriate modification to the expression for the perpendicular case. Limitations in the calculation of impact angle from the stain aspect ratio are identified and a theoretical basis for the prediction of spines around an elliptical stain is proposed. Some key issues for future research are identified which include a systematic, quantitative study of the effect of surface properties on bloodstain formation.     

Application of Volume‐of‐Fluid Method to Analyze the Viscosity Effect on the Spine Formation of Bloodstains

In bloodstain pattern analysis, the blood droplet volume and surface impact velocity play an important role, and many related experimental studies have been carried out. If an appropriate computational fluid dynamics (CFD) model that could solve bloodstain patterns, especially spine formation bloodstain patterns, can be obtained, the blood droplet volume and impact speeds at various crime scenes can be predicted more accurately. For this purpose, Flow‐3D software using the volume‐of‐fluid method was applied to analyze the behavior of human blood droplets during an impact event, especially focusing on the viscous effect on splashing, which forms the spine which can be used to predict the impact velocity. To obtain a non‐Newtonian viscosity model of blood for a computational fluid dynamic analysis, the venous blood samples of 163 people were tested using a hemorheology instrument. Among the venous blood samples of 163 people, 37 samples for which all blood test results were in a normal range were selected for the non‐Newtonian viscosity modeling. From the CFD analysis, it could be concluded that a non‐Newtonian viscosity model is more appropriate than a constant viscosity model for predicting splashing that forms the spine. The gradient of the non‐Newtonian model at a high shear rate has more of an effect on spine formation than that at a low shear rate. The lowest viscosity with a high velocity at the outer front of the radiating flow plays an important role in forming the splashing pattern.     

Distinction of bloodstain patterns from fly artifacts

Forensic scientists may encounter blood spatter at a scene which may be pure or a mixture of fly artifacts and human bloodstains. It is important to be able to make an informed identification, or at least advanced documentation of such stains since the mechanics of production of fly artifacts are not determinable to the crime scene reconstructionist from regular police forces. We describe three cases in which experiments and crime scene reconstruction led to additional information. Case 1: Above the position of a victim, numerous blood stains of the low-high-velocity type were found. Exclusion of these stains being caused by force (but instead caused by the activity of adult blow flies) by use of the following observations that were confirmed in experiments: (a) sperm-/tadpole-like structure with length > width, (b) random directionality, and (c) mixture of round symmetrical and teardrop shaped stains. Case 2: A reddish spatter field was found on a fan chain two rooms away from the place where a dead woman was found. Localization of the spatter on the bottom end of the surface hinted strongly towards fly activity. Case 3: Double homicide; submillimeter stains were found on a lamp between the two corpses. Activity of flies was less likely compared to alternative scenario of moving lampshade and violent stabbing.