The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle–Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt.     

Bloodstains on Leather: Examination of False Negatives in Presumptive Test and Human Hemoglobin Test

Presumptive tests for blood are very simple and sensitive tests used in the search for evidence. They also provide initial information on the nature of stains. A second test can confirm their nature. However, these tests can present false–negative results for different reasons. Some of those reasons have been studied, while others, those caused by the substrate material that contains the stain, are less well known. This work studies the effect of one component of a leather substrate—quebracho extract—on presumptive and human hemoglobin blood tests. Assays were performed using samples of blood dilutions contaminated with quebracho extract and others formed on a substrate containing the contaminant. Results show an undoubted interference that causes false negatives and even visible to the naked eye stains and also indicate that some tests (phenolphthalein) are more affected than others. Examiners should be taken into account when working on this kind of substrates.     

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.     

The Strategic Use of Legislative Voting Procedures

Legislative votes can be taken by roll call—noting the position of each individual member—or by some form of indication (sitting or standing, shouting yea or nay, etc.)—noting only an aggregate outcome. Cameral rules define one method of voting as the standard operating procedure and how to invoke any alternative voting methods. We develop a series of hypotheses related to position taking to explain why, when procedures would typically lead to a vote taken by indication, legislators choose to vote by roll call—a means that makes it much easier for actors outside the chamber to observe the positions taken by individual legislators and partisan blocs. With data from Argentina and Mexico, we test these hypotheses regarding the strategic choice of vote procedures and their relationship to observed party unity.     

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.     

Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy

Non-destructive identification and subsequent age estimation of blood stains are significant steps in forensic casework. The latter can provide important information on the temporal aspects of a crime. As previously shown, visible spectroscopy of blood stains on white backgrounds can successfully be used for their identification and age estimation. The use of this technique however, is hampered by dark backgrounds. In the present study the feasibility to use near infrared (NIR) spectroscopy was evaluated for blood stain identification and age estimation on dark backgrounds. Using NIR reflectance spectroscopy, blood stains were distinguished from other substances with 100% sensitivity and 100% specificity. In addition, Partial Least Squares Regression analysis was applied to estimate the age of blood stains on colored backgrounds. The age of blood stains up to 1 month old was estimated successfully with a root mean squared error of prediction of 8.9%. These findings are an important step toward the practical implementation of blood stain identification and age estimation in forensic casework, where a large variety of backgrounds can be encountered.     

Assessment of shooting distance on the basis of bloodstain analysis and histological examinations

A 28-year-old man was shot using a pump-gun. The main question to be resolved was whether the biological stain pattern on the suspect's trousers, and in particular the bloodstains, can provide evidence to assess the shooting distance between the suspect and the position of the victim's body. The biological stain pattern (i.e. bloodstains and brain tissue) showed backspatters from the shot entrance wound on the back of the head, while the victim was lying face down and the suspect was standing close behind his head.     

Site, number and depth of wounds of the scalp in falls and blows--a contribution to the validity of the so-called hat brim rule

The sites, numbers and lengths of wounds of the skin of the scalp and head caused by blunt injuries (falls from standing position, falls downstairs, blows) in an autopsy series. Analysis of the localization, length and number of wounds located at the head in 305 autopsy cases of falls from a standing position (203 cases), falls downstairs (51 cases) and blows with relatively long materials (51 cases). For the distinction between falls and blows, among several other aspects the so-called rule of the hat-brim-line is described: in cases of falls from a standing position down to a flat bottom, contusions can be expected in or near a line which represents the greatest horizontal circumference of the skull, whereas in cases of blows the contusions lay above this area. Although such a tendency was found in our material, a rule could not be confirmed: only approximately 55% of the wound due to blows were above this line, and in cases of falls from a standing position ca. 1/3 of the wounds were above this area. Only in the (dorso-frontal and parietal) region "top of the head" contusions due to falls from a standing position were quite uncommon. The mean length and length distribution of wounds in the hat-brim-zone was not different between the 3 causes of injuries; in positions above this, wounds due to blows were longer compared to those by falls. In falls from a standing position, only exceptionally more than 1 wound was found, whereas in cases of homicide several or many wounds were common. Single wounds due to blows were only found in cases, in which the victims were knocked down but not killed by blunt forces, and death was caused by other methods (e.g. strangulation or stabbing).     

The analysis of biological samples from crime scene for a future human DNA profile confrontation. Effects of presumptive test reagents on the ability to obtain STR profiles for human identification

Because of the increase of evidence of blood stains, that have been washed or cleaned in an attempt to mask the analysis of DNA profiles, there is also an increase in the use of presumptive tests on samples sent to laboratories. Some of the presumptive tests, used to identify blood and semen stains, could potentially affect the recovery of high molecular weight DNA from the samples, or extinguish them, especially those already present in small quantities. After the presumptive tests, often these samples are discarded. This study aimed to examine the possibility of obtaining a DNA profile from samples submitted for presumptive testing and cleaned with bleaches with and without chlorine. Two different protocols were conducted: (a) A unique sample of human blood in natura (5 μL), already typed through the DNA techniques with the genetic profile previously known (control), was distributed onto cotton fabrics and dried at room temperature. Four samples of fabric were macerated in saline solution and Coombs serum and then stored for three months (room temperature and freezer −20 °C). (b) Another sample of human blood, type A, in natura, already typed through the techniques of DNA (control) was used. Aliquots of 200 μL were distributed in: cotton, denim and synthetic fabric. The samples were dried at room temperature for 24 h. The blood stains in those fabrics (cotton, denim and synthetic) were then divided into three groups: unwashed, cleaned with chlorine bleach and cleaned with chlorine bleach and soap powder. The samples were again dried at room temperature for 24 h, before the use of luminol. The DNA were extracted with Chelex 100 and amplified with the Identifiler Kit (Applied Biosystems). The blood stains exposed to saline and Coombs serum had DNA profiles consistent with untreated samples (controls). This result shows that the experts should keep and store the samples treated with saline and Coombs serum for future DNA confrontation when necessary. Also discussed in this paper the pattern of blood stains after washing with bleaching solutions, as well as the quantity of DNA obtained from these samples.     

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.     

Thoracic Visceral Compression–A Useful Technique for Demonstrating Small Bleeding Points in the Dissected Neck at Autopsy

A 29-year-old man died at the scene of an altercation from a stab wound to the left side of the neck. At autopsy, careful in situ examination of the common carotid artery did not reveal any evidence of hemorrhage or apparent vascular injury. However, applying pressure to the chest wall and underlying thoracic viscera (lungs and heart) resulted in filling of the collapsed vessel with blood causing hemorrhage from a small incision on the medial aspect of the common carotid artery. Release of pressure and drying of the dissection field enabled the artery to be opened and the small defect to be identified. This technique may be used to temporarily restore blood to a vessel to help identify a small bleeding point that may otherwise be difficult to detect.     

A Molecular Method to Detect Wound Cells in Bloodstains Resultant of Sharp Force Injuries for Crime Scene Reconstruction

Previous research by the authors on an animal model showed that bloodstains can contain additional information about their somatic origin in the form of wound cells. Bloodstains produced by a gunshot wound to the head were distinguished from bloodstains produced by a gunshot wound to the chest by testing the stains for a brain microRNA marker. In this study, the effectiveness of the technique was examined on blood drops shed externally from a stab wound to the liver of rat carcasses. Specifically, investigations were conducted on the liver microRNA marker, rno‐mir‐122‐3p, with the QIAGEN miScript System, and PCR analysis. Between the two stabbing methods used, 67% of the scalpel blades and 57% of the blood drops tested positive for rno‐mir‐122‐3p; however, other samples tested negative giving inconclusive results as to the wound‐of‐origin. The amount of the liver cells in the bloodstains appeared to be related to the extent of trauma.     

Estimating age of humans based on telomere shortening

To estimate age using DNA based on telomere shortening, we determined the terminal restriction fragment (TRF) length, as telomere length, using Southern blot analysis of peripheral human blood and blood stains. All blood stains had been stored at room temperature for 5 months. The average TRF length clearly showed a tendency to shortening with aging. The formula for age estimation was based on a correlation between average TRF length and age of the subjects. The estimated age calculated from TRF length widely depends on environmental and genetic factors. However, as long as the DNA is well preserved, use of our method is feasible regardless of age of the subject and can give a rough estimation of age of subjects in forensic samples that carry no morphological information.     

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.     

Fatal hemorrhage from a periumbilical wound: Stabbing or hemorrhage from a caput medusae?

Varices are the main clinical manifestation of portal hypertension, and their bleeding is the predominant cause of mortality from this condition. Periumbilical varices are known as “caput medusae.” Reports of their bleeding are rare, with only three fatal cases described in the literature. The antemortem diagnosis is relatively simple, while the postmortem diagnosis is more complex. This paper is the first report of fatal hemorrhage from a caput medusae for which the diagnosis was made postmortem, thanks to a complete diagnostic process including scene and circumstances, medical history, and autopsy with detailed histology. The circumstantial analysis showed the presence of a large amount of blood at the scene, blood which originated from a small abdominal wound; an analysis of the subject's clinical data reported that he was affected by portal hypertension. The autopsy revealed some dilated and convoluted veins in the subcutaneous tissue of the umbilical region; a fistula between these veins and the abdominal wound was detected. The histological study confirmed the presence of periumbilical varices, one of them ruptured and connected with the overlying skin. The cause of death was attributed to a massive hemorrhage generated by a periumbilical varix in a patient affected by portal hypertension.     

The Reliability of Pattern Classification in Bloodstain Pattern Analysis—PART 2: Bloodstain Patterns on Fabric Surfaces,

This study was designed to produce the first baseline measure of the reliability of bloodstain pattern classifications on fabric surfaces. Experienced bloodstain pattern analysts classified bloodstain patterns on pairs of trousers that represented three fabric substrates. Patterns also varied in type (impact, cast‐off, expiration, satellite stains from dripped blood, and transfer) and extent. In addition, case summaries that accompanied each pattern contained contextual cues that either supported the correct answer (i.e., positive bias), were misleading toward an incorrect answer (i.e., negative bias), or contained no directional information (i.e., neutral). Overall, 23% percent of the resulting classifications were erroneous. The majority (51%) of errors resulted from analysts misclassifying satellite stains from dripped blood. Relative to the neutral information, the positive‐bias information increased correct classifications and decreased erroneous classifications, and the negative‐bias information decreased correct classifications and increased erroneous classifications. The implications of these findings for BPA are discussed.     

A Molecular Method to Correlate Bloodstains with Wound Site for Crime Scene Reconstruction

Bloodstain pattern analysis to determine the wound‐of‐origin of bloodstains is problematic with nonspecific patterns. In this proof‐of‐concept study, the authors examined a molecular approach to correlate bloodstains with injuries using the rat as a model. Specifically, investigations were conducted on the rat brain marker, rno‐miR‐124‐3p, with the QIAGEN miScript System and real‐time PCR analysis. Rno‐miR‐124‐3p was detected in brain homogenates diluted 100,000 times; in 3‐week‐old, room temperature stored, simulated brain–blood stains; and in bloodstains from head gunshot wounds collected with swabs and subsequently frozen for 9–18 months; however, rno‐miR‐124‐3p was not detected in whole blood. Proof‐of‐principle was demonstrated by the ability to distinguish bloodstains from a gunshot wound to the head versus bloodstains from a gunshot wound to the chest, by the testing of otherwise identical bloodstains from the two patterns for the presence of the marker. The results suggest a viable approach to a longstanding problem in casework.     

Alcohol‐Induced Blackout as a Criminal Defense or Mitigating Factor: An Evidence‐Based Review and Admissibility as Scientific Evidence

Alcohol‐related amnesia—alcohol blackout—is a common claim of criminal defendants. The generally held belief is that during an alcohol blackout, other cognitive functioning is severely impaired or absent. The presentation of alcohol blackout as scientific evidence in court requires that the science meets legal reliability standards (Frye, FRE702/Daubert). To determine whether “alcohol blackout” meets these standards, an evidence‐based analysis of published scientific studies was conducted. A total of 26 empirical studies were identified including nine in which an alcohol blackout was induced and directly observed. No objective or scientific method to verify the presence of an alcoholic blackout while it is occurring or to confirm its presence retrospectively was identified. Only short‐term memory is impaired and other cognitive functions—planning, attention, and social skills—are not impaired. Alcoholic blackouts would not appear to meet standards for scientific evidence and should not be admissible.     

Predicting the position of the source of blood stains for angled impacts

Droplets of pig's blood were dropped onto paper at different angles to the horizontal to produce blood stains. Impact velocities varied from 1.82 to 5.76 m/sec, drop size from 3.7 to 5.0 mm in diameter, and the surface sloped at angles between 22.7 degrees and 90 degrees to the horizontal. From the data a single equation relating stain size to drop size and velocity for all impact angles was produced; ab = 111.74 (Re(1/2)We(1/4))(0.75)D(o)D(o) + 0.00084 with R(2) = 0.88, where a is the stain width, b the stain length, Re the Reynolds number, and We the Weber number. A second equation related the number of spines, N, to drop size, velocity, and surface slope for all impact angles as N = 0.76 We(0.5) sin(3)theta with R(2) = 0.9, where theta is the impact angle. Combining these equations the impact velocity can be determined and hence the position of the stain's source.