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.     

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.     

用国产两性电解质进行血痕的种属鉴定

本文应用国产两性电解质等电点聚焦法(PAGIEF)对1个月的40种动物血痕浸出液及经PCMB处理后的血痕浸出液的谱型进行了研究。根据谱型特征,成人除与人胎儿及猴难以鉴别外,与其它动物均可鉴别。动物间除鸟纲,鱼纲内有五组动物组内难以鉴别外,其它均能鉴别。经PCMB处理后,成人与人胎儿的谱型仍无差别,与其它动物的鉴别则更容易。结果表明,国产两性电解质用于等电聚焦进行血痕的种属鉴别是完全可能的。     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes. The use of a versatile light source may assist in their detection. The ability of Polilight to locate potential semen, saliva, and blood stains on a range of substrates and at different dilutions was tested. We also tested the use of Polilight in comparison with conventional chemical-based presumptive screening tests such as acid phosphatase (AP), Phadebas, and luminol, often used in casework for detecting potential semen, saliva, and blood stains, respectively. The Polilight was able to locate stains that were not apparent to the naked eye. The color of the material on which a stain is deposited can have an effect on the detectibility of the stain. The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity. In a comparative study between the AP test and Polilight on 40 casework exhibits, one false-negative result was observed when using the Polilight. On a series of mock casework exhibits it was determined that the Polilight can be used successfully to locate saliva stains for DNA analysis. The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight; however the Polilight has particular application in instances where a bloodstain may have been concealed with paint. Overall, the Polilight is a relatively safe, simple, noninvasive, and nondestructive technique suitable for use in forensic casework.     

The Reliability of Swipe/Wipe Classification and Directionality Determination Methods in Bloodstain Pattern Analysis

Swipe and wipe are types of transfer patterns commonly encountered at bloodied scenes. So far, there have been little published data on the reliability of evaluating these patterns. In this study, 36 bloodstain patterns were randomly assigned to 12 analysts in three separate trials, and the analysts were required to evaluate the pattern type and directionality. The results revealed that correct classifications of patterns type were problematic, with an overall error rate of 32%. Wipes created from wet bloodstains were the most difficult to identify due to the absence of signs of alteration. The directionality of swipes made with a gloved finger had a 100% success rate; however, for swipes made with cloth, the analysts mistook the direction in nearly every case, which is of significant concern, considering these are common patterns at crime scenes. This study suggested there is a need for revising the current protocols for interpretation of these pattern types.     

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.     

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.     

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.     

Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions.

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.     

A new peptidase isozyme which may assist in the identification of vaginal debris

A characteristic peptidase has been identified in vaginal swab extracts. This enzyme, which has been termed vaginal peptidase, can be identified by its high anodal electrophoretic mobility in starch gel at pH 7.4 and its ability to hydrolyse the dipeptide substrate L-valyl-L-leucine. Vaginal peptidase was not detected in any of the body fluids or excretions commonly encountered in forensic stain analysis, other than vaginal debris. The use of this enzyme as a biochemical marker for the identification of vaginal debris in dried stains is discussed.     

Exsanguinated blood volume estimation using fractal analysis of digital images

The estimation of bloodstain volume using fractal analysis of digital images of passive blood stains is presented. Binary digital photos of bloodstains of known volumes (ranging from 1 to 7 mL), dispersed in a defined area, were subjected to image analysis using FracLac V. 2.0 for ImageJ. The box-counting method was used to generate a fractal dimension for each trial. A positive correlation between the generated fractal number and the volume of blood was found (R(2) = 0.99). Regression equations were produced to estimate the volume of blood in blind trials. An error rate ranging from 78% for 1 mL to 7% for 6 mL demonstrated that as the volume increases so does the accuracy of the volume estimation. This method used in the preliminary study proved that bloodstain patterns may be deconstructed into mathematical parameters, thus removing the subjective element inherent in other methods of volume estimation.     

The detection of Y chromosomes in bloodstains--a reevaluation

A method has been described for detecting Y chromosomes in the leukocytes of human bloodstains prepared on a variety of substrates. The factors that influence the proportion of chromosomes exhibiting a Y spot (the Y cell index) in a bloodstain are considered, including the subjective nature of assessment of the Y chromosome fluorescence, the substrate, and the age of bloodstain. In contrast to previous workers no decay in Y cell index with the age of the stain was observed. The results of a blind trial involving stains derived from case work, where from other evidence there was no doubt as to the sex of the donor, are presented. Sixty-five percent of the male bloodstains were correctly identified and no females were wrongly reported as male.     

Expert opinion on biologic stains. Determination of status, future trends

In this paper an attempt is made to critically review the literature, with special emphasis on bloodstain analysis. One essential aim is the integration of this field into casework. Three basic components in skillful assessment of stains are described: (1) analysis of stain morphology, (2) discriminating and attributing analyses, (3) individualization. Regarding the first, the analysis of stain morphology is based upon the extensive experimental literature published since 1895--mainly in continental Europe. Since 1971 there have also been publications in the American literature. The large family of stain forms and their dependency on multiple variables are described, especially regarding the modes of formation, the energy of impact, and the physical properties of the substrate. The essential elements for reconstruction of the crime are described. The areas of application are arranged in case groups. Since in case work the stain pattern is complicated by many artifacts and overlaps, forensic pathologists are considered the ideal experts for the analysis of bloodstain patterns, as they have a profound knowledge of the type and sequence of injuries. If this is not the case, the forensic pathologist should at least be integrated into the investigating team. In practical application, the stain form is not always adequately analysed. The education and training of pathologists should be improved to achieve this standard. Analysis of the stain morphology and a subsequent selection of stains are also essential prerequisites for meaningful further investigations. By the use of discriminating and attributing analyses, one can as a rule arrive at a definite answer by using only one test. This is true for basic questions such as the identification of blood type, as well as proof of exclusion. One can distinguish between traditional methods, the new field of immunochemistry and rarely used methods. Immunochemistry has permitted success in recent years in determination of the blood group from hair. It is recommended that reference laboratories be established for training in these rare methods. Individualization analyses are subdivided into two large fields: non-DNA individualization and DNA individualization. It is postulated that in the future stain laboratory both areas will coexist. In non-DNA individualization, essential progress has been made. The detection of protein polymorphisms by blotting and subsequent visualization by antibody-linked enzyme/substrate reactions has led to a considerable increase in sensitivity and specificity.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of fabric surface characteristics on satellite bloodstain morphology

Bloodstains on fabrics such as clothing, soft furnishings or carpets are often encountered in casework. These stains often have a distinctive morphology that includes satellite stains, thought to be a highly sensitive feature that is a function of surface roughness. This study presents the findings of experimental studies conducted with proxy blood on two fabrics, similar in labeled composition, to assess the influence of fabric type on satellite stain generation. The morphology of proxy blood stains on the two fabric types were found to be statistically distinguishable from one another, with the volume of satellite stains generated being dependent upon the surface roughness of the fabric. These findings provide an initial step that illustrates the viability of providing an empirical evidence base for the interpretation of satellite stains in forensic blood pattern analysis (BPA).     

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.     

Identification in blood stains through DNA typing with C4 and HLA-DR probes

A restriction fragment length polymorphism analysis using double digestion of DNA preparations with XbaI and BglII restriction enzymes and hybridization with C4 and HLA-DR probes is described. The typing conditions selected reveal extensive individual variation in both C4 and DR gene regions. In our panel of 46 unrelated individuals, 37 different phenotypic patterns were recognized when both probes were used, and preliminary discriminative power values of 0.865 and 0.914 were calculated for C4 and DR beta, respectively. The probability of a chance match using both systems is probably about 1.5.10(-2). The potential of this method for individual identification of blood stains was demonstrated on DNA prepared from 6-month-old dried blood stains from seven panel individuals. The seven individuals were all identified when comparing stain DNA patterns with panel control patterns. No RFLP pattern changes were observed following storage of blood stains. Based on these experiments with C4 and DR beta DNA typing under laboratory conditions, it is concluded that DNA typing with such probes may become a powerful tool in future stain identification analyses.     

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.     

A new marker for estimation of bloodstain age by high performance liquid chromatography.

The applicability of a new marker for estimation of bloodstain age by reverse-phase high performance liquid chromatography (HPLC) is described. Using a microBondasphere C18 column with a two step linear gradient of 10.5-46.25% acetonitrile in 0.1% trifluoroacetic acid, an intriguing peak (unidentified) at a retention time of about 5 min was observed on chromatograms from human adult bloodstains and designated as 'X'. The area of this peak, which could be detected in extracts of bloodstains, but not in their fresh whole blood, increased with time. The ratios of the X area to heme area in bloodstains stored at room temperature and 4 degrees C for up to 52 weeks old linearly correlated with stain age by plotting on a double logarithmic scale. In bloodstains exposed to fluorescent light at room temperature, the regression equation calculated from the ratios (Rx) and the ages of stains in weeks (W) is ln(1000.Rx) = 1.1084 + 0.3937.ln(7.W), and the coefficient of correlation (r) is 0.9776 (n = 144, P < 0.001). When stains were stored at 37 degrees C, the ratio transformed into logarithms correlated linearly with stain age. The regression equation describing the relationship in bloodstains exposed to fluorescent light at 37 degrees C is ln(1000.Rx) = 2.4477 + 0.0866.W (r = 0.9826, n = 144, P < 0.001). The results of the present study suggest that the HPLC method may be applicable to the estimation of bloodstain age.     

Sex identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic acid (DNA) analyzing technique

Deoxyribonucleic acid (DNA) specimens were prepared from blood or bloodstain extracts, and the content of a Y-chromosome specific DNA fragment was investigated by the Southern hybridization method using a nonisotopic staining technique. Thus obtained patterns of male DNA showed a clear band, whereas broad stains with some faint bands appeared on the patterns of DNA from both sexes. This method is expected to be a new powerful mean of forensic medical examination.     

Chemical Enhancement Techniques of Bloodstain Patterns and DNA Recovery After Fire Exposure*

Abstract: It is common in forensic casework to encounter situations where the suspect has set a fire to cover up or destroy possible evidence. While bloodstain pattern interpretation, chemical enhancement of blood, and recovery of deoxyribonucleic acid (DNA) from bloodstains is well documented in the literature, very little information is known about the effects of heat or fire on these types of examinations. In this study, a variety of known types of bloodstain patterns were created in a four‐room structure containing typical household objects and furnishings. The structure was allowed to burn to flashover and then it was extinguished by firefighters using water. Once the structure cooled over night, the interior was examined using a bright light. The bloodstains were evaluated to see if the heat or fire had caused any changes to the patterns that would inhibit interpretation. Bloodstain patterns remained visible and intact inside the structure and on furnishings unless the surface that held the blood was totally burned away. Additionally, a variety of chemical techniques were utilized to better visualize the patterns and determine the possible presence of blood after the fire. The soot from the fire formed a physical barrier that initially interfered with chemical enhancement of blood. However, when the soot was removed using water or alcohol, the chemicals used, fluorescein, luminol, Bluestar^®, and Hemastix^®, performed adequately in most of the tests. Prior to DNA testing, the combined phenolphthalein/tetramethyl benzidine presumptive test for the presence of blood was conducted in the laboratory on samples recovered from the structure in an effort to assess the effectiveness of using this type of testing as a screening tool. Test results demonstrated that reliance on obtaining a positive presumptive result for blood before proceeding with DNA testing could result in the failure to obtain useful typing results. Finally, two DNA recovery methods (swabbing the stain plus cutting or scraping the stain) were attempted to evaluate their performance in recovering samples in an arson investigation. Recovery of DNA was more successful in some instances with the swabbing method, and in other instances with the cutting/scraping method. Therefore, it is recommended that both methods be used. For the most part, the recovered DNA seemed to be unaffected by the heat, until the temperature was 800°C or greater. At this temperature, no DNA profiles were obtained.