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.     

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.     

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.     

The Food Preferences of the Blow Fly Lucilia cuprina Offered Human Blood,Semen and Saliva,and Various Nonhuman Foods Sources

As human DNA profiles can be obtained from blow fly artifacts, this study aimed to establish the feeding preferences of Lucilia cuprina (Wiedemann) blow flies when offered human biological fluids and nonhuman food sources. One‐day‐old and 3‐day‐old blow flies of both sexes were simultaneously offered human blood, semen and saliva, pet food, canned tuna and honey, and the number and length of visits documented over 6 h. One‐day‐old flies visited pet food and honey most often, but stayed longest on honey and semen. Three‐day‐old flies visited semen and pet food most often, and stayed longest on these food sources. Blood and saliva were the least preferred options for all flies. Overall, flies preferred dry blood and semen to the wet forms. These findings demonstrate that even when other food sources are available, flies at a crime scene may feed on human biological fluids if present, potentially transferring human DNA.     

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.     

Location of Artifacts Deposited by the Blow Fly Lucilia cuprina After Feeding on Human Blood at Simulated Indoor Crime Scenes

Human DNA profiles can be obtained from fly artifacts (feces and regurgitant) when a fly has been feeding on biological material, sometimes 2 years after deposition. Morphological similarity between artifacts and spots of unaltered biological material make it difficult to distinguish between them, and presumptive and confirmatory forensic tests are unreliable in making the distinction. Knowing possible artifact locations will assist investigators in recognizing where DNA contamination might occur. Flies were released into a house with human blood available under a variety of different climatic and lighting conditions. The location of flies and artifacts was recorded after 72 h. It was found flies may move toward warm or well‐lit areas and deposit artifacts there, but artifacts were predominantly located around food sources and were often found in low positions. Factors such as ambient temperature, and the proximity of light and food sources, had an impact on where artifacts were deposited.     

Blood-spatter patterns: hands hold clues for the forensic reconstruction of the sequence of events

Biologic and nonbiologic traces on the hands are of particular importance for the forensic reconstruction of shooting incidents; gunpowder residue analysis in particular helps determine whether the gunshot is close range or distant. In crime scene investigation, knowledge about the morphology of bloodstain patterns-including gunshot-related back spatter-has increased since various experimental examinations have been performed in the last years; nowadays, these traces are frequently used for forensic crime scene reconstruction. The goal of this study was to deduce the position and orientation of the hands, and therefore the firearm, according to the bloodstain patterns on the hands of the deceased. For this purpose blood-spatter stains on the hands were examined on site in 5 suicides caused by gunshot. In all cases, forensically relevant conclusions regarding forensic reconstruction were enabled through close examination of the spatter marks. Therefore, in shooting incidents, analysis and documentation of blood-spatter findings on the hands is recommended before hands are tested for gunpowder residue or wrapped for the transport of the body.     

Automatic Classification of Bloodstain Patterns Caused by Gunshot and Blunt Impact at Various Distances

The forensics discipline of bloodstain pattern analysis plays an important role in crime scene analysis and reconstruction. One reconstruction question is whether the blood has been spattered via gunshot or blunt impact such as beating or stabbing. This paper proposes an automated framework to classify bloodstain spatter patterns generated under controlled conditions into either gunshot or blunt impact classes. Classification is performed using machine learning. The study is performed with 94 blood spatter patterns which are available as public data sets, designs a set of features with possible relevance to classification, and uses the random forests method to rank the most useful features and perform classification. The study shows that classification accuracy decreases with the increasing distance between the target surface collecting the stains and the blood source. Based on the data set used in this study, the model achieves 99% accuracy in classifying spatter patterns at distances of 30 cm, 93% accuracy at distances of 60 cm, and 86% accuracy at distances of 120 cm. Results with 10 additional backspatter patterns also show that the presence of muzzle gases can reduce classification accuracy.     

Morphological Features of Regurgitate and Defecatory Stains Deposited by Five Species of Necrophagous Flies are Influenced by Adult Diets and Body Size

The morphological characteristics of artifacts from five species of necrophagous flies were examined following feeding on several types of diets. Four types of insect stains were produced by each species: regurgitate, defecatory, translocation, and tarsal tracks. Regurgitate was the most frequent type deposited (70.9 ± 2.4%), followed by defecatory (19.8 ± 4.0%), tarsal tracks (8.6 ± 1.2%), and translocation (0.7 ± 0.1%). Artifact shapes, sizes, and color were highly variable and species and diet specific. Calliphora vicina and Sarcophaga bullata consistently deposited the largest artifacts after feeding, whereas Chrysomya rufifacies and Ch. megacephala produced more tarsal tracks than the other species examined. Artifacts with tails were infrequently observed (4.1 ± 0.6% of all stains) but occurred as either defecatory or regurgitate stains. The widely variable morphologies of all types of fly artifacts underscores the view that insect stains cannot be distinguished from human bloodstains based on morphology alone.     

Forensic entomology exemplified by a homicide. A combined stain and postmortem time analysis]

The combined analysis of both ant and blow fly evidence recovered from a corpse, and from the boot of a suspect, suggested that an assumed scenario in a high profile murder case was likely to be true. The ants (Lasius fuliginous) were used as classical crime scene stains that linked the suspect to the scene. Blow fly maggots (Calliphora spec.) helped to determine the post mortem interval (PMI) with the calculated PMI overlapping with the assumed time of the killing. In the trial, the results of the medico-legal analysis of the insects was understood to be crucial scientific evidence, and the suspect was sentenced to 8 years in prison.     

“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.     

Quantitative Differentiation of Bloodstain Patterns Resulting from Gunshot and Blunt Force Impacts

Bloodstain pattern analysis (BPA) provides significant evidentiary value in crime scene interpretation and reconstruction. In this work, we develop a quantitative methodology using digital image analysis techniques to differentiate impact bloodstain patterns. The bloodstain patterns were digitally imaged and analyzed using image analysis algorithms. Our analysis of 72 unique bloodstain patterns, comprising more than 490,000 individual droplet stains, indicates that the mean drop size in a gunshot spatter pattern is at most 30% smaller than the mean drop stain size in blunt instrument patterns. In contrast, we demonstrate that the spatial distribution of the droplet stains—their density as a function of position in the pattern—significantly differs between gunshot and blunt instrument patterns, with densities as much as 400% larger for gunshot impacts. Thus, quantitative metrics involving the spatial distribution of droplet stains within a bloodstain pattern can be useful for objective differentiation between blunt instrument and gunshot bloodstain patterns.     

Visualization of Latent Blood Stains Using Visible Reflectance Hyperspectral Imaging and Chemometrics

The detection of latent traces is an important aspect of crime scene investigation. Blood stains on black backgrounds can be visualized using chemiluminescence, which is invasive and requires a darkened room, or near-infrared photography, for which investigators need to change filters manually to optimize contrast. We demonstrated the performance of visible reflectance hyperspectral imaging (400–720 nm) for this purpose. Several processing methods were evaluated: single wavelength bands, ratio images, principal component analysis (PCA), and “SIMPLe-to-use Interactive Self-modeling Mixture Analysis” (SIMPLISMA). Using these methods, we were able to enhance the contrast between blood stains and 12 different fabrics. On black cotton, blood dilutions were visible with a minimal concentration of 25% of whole blood. The hyperspectral camera system used in this study is portable and wireless, which makes it suitable for crime scene use. The described technique is noncontact and nondestructive, so all traces are preserved for further analysis.     

Comparison of Four Saliva Detection Methods to Identify Expectorated Blood Spatter

Blood spatter analysis is an important step for crime scene reconstruction. The presence of saliva in blood spatter could indicate expectorated blood which is difficult to distinguish from impact spatter. In this study, four saliva test methods (SALIgAE^®, Phadebas^® sheet, RSID^™-Saliva kit, and starch gel diffusion) were compared to identify the best method for detecting expectorated blood spatter. The RSID^™-Saliva kit showed the highest sensitivity even when saliva was mixed with blood, and was not inhibited by the presence of blood. The SALIgAE^® test provided easy and rapid results, but the yellow color of a positive reaction was overwhelmed by the red color of the blood. The starch gel diffusion method and the Phadebas^® sheet exhibited relatively low sensitivity and the assay took a long time. When using the RSID^™-Saliva kit for identifying saliva in blood, results should be read within 10 min.     

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.     

Detection and visualization of human tears using alternate light sources for forensic purposes

The current scientific techniques for locating body fluids focus on quick and effective methodologies for easy and reliable identification. Efficient detection and identification of body fluids play a vital role in establishing the ‘corpus delecti’ of a crime. Non-destructive techniques such as the use of Alternate Light Sources (ALS) have been exploited for crime scene searches over large areas and detection of body fluids such as blood, semen, vaginal secretions, and saliva on a range of substrates. Tears are rarely found but can be considered as potential body fluid evidence due to their unique biochemical and molecular properties. Tears are secreted in response to physical or emotional stimuli. Due to the small volume of secretions, they are often overlooked in the crime scene. Tears may be found on surfaces such as clothing, bedding, tissue, handkerchief, or balaclava. The use of ALS to locate tears on tissue paper and fabric surfaces was tested which were not apparent to the naked eye. Tears stains were successfully detected on surfaces of forensic interest with varying sample ages up to three months with a broad excitation spectrum between 254 nm and 410 nm. Dried stains on tissue paper and fabric substrates were better detected with sharp margins, clear stain pattern visibility, and fluorescence intensity in comparison with moist and fresh stains. Tears stains can hence be detected with the use of ALS and suitable filter combinations under normal conditions and do not require any specific settings to locate them. These findings are suggestive for easy and quick identification of tears on large surfaces and as a presumptive test for forensic casework evidence examination.     

Impact Spatter Bloodstain Patterns on Textiles

There are few reports of studies of impact spatter on textiles even though bloodstained textiles are found at many violent scenes. Impact spatter was deposited at 90° impact angle onto three knit fabrics of different yarn sizes and on paper. The resulting stain areas and number of stains were measured using ImageJ and compared with stains on paper using one‐factor ANOVA. The number of stains observed and their areas on the knit fabrics decreased as the yarn size increased. It was also found that blood that deposited on the fabric wicked only in the direction of the fibers at that location within the fabric which led to distorted stain shapes. Fewer observed impact spatter stains were found on cotton jersey knits for fabrics made with larger yarns than on paper. As the yarn size became smaller, the number of stains became the same as on paper.     

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.     

An evaluation of the underlying mechanisms of bloodstain pattern analysis error

An experiment was designed to explore the underlying mechanisms of blood disintegration and its subsequent effect on area of origin (AO) calculations. Blood spatter patterns were created through the controlled application of pressurized air (20-80 kPa) for 0.1 msec onto suspended blood droplets (2.7-3.2 mm diameter). The resulting disintegration process was captured using high-speed photography. Straight-line triangulation resulted in a 50% height overestimation, whereas using the lowest calculated height for each spatter pattern reduced this error to 8%. Incorporation of projectile motion resulted in a 28% height underestimation. The AO xy-coordinate was found to be very accurate with a maximum offset of only 4 mm, while AO size calculations were found to be two- to fivefold greater than expected. Subsequently, reverse triangulation analysis revealed the rotational offset for 26% of stains could not be attributed to measurement error, suggesting that some portion of error is inherent in the disintegration process.     

Observations and 3D Analysis of Controlled Cast-Off Stains

Cast-off stains are common patterns found at crime scenes involving blood shedding events. However, the analysis and interpretation of cast-off patterns remains an area lacking tools for crime scene investigators. Analyzing cast-off patterns may allow investigators to interpret the area from where an object may have been swung and thus determine the approximate location of a suspect or victim. This study looked at the position and distribution of cast-off patterns and area of origin as a starting point for the development of a method to analyze cast-off patterns. Through a series of tests using a controlled cast-off rig (n = 10), it was observed that a Path Volume Envelope (PVE) may be identified where the swinging path is contained in a volume along with an area of exclusion. The calculated center, linear position of the PVE was found to have an average error of just over 3.2 cm when compared to the known object swing path position. The maximum deviation of the PVE to the known swing path was found to be 5.0 cm with a standard deviation of 1.4 cm. Additional studies are required to investigate the effects of partial cast-off stains, wielded object velocity, direction of swing, distance from the projected surface, and other factors. The observations and analysis from this study were seen to be predictable and repeatable and may provide a possible new method for investigators to interpret cast-off stains.