Chemistry in crime investigation: sodium percarbonate effects on bloodstains detection

Chemistry plays a leading role in crime investigation. In the study of bloodstains, chemical reactions provide the means for the detection. All these procedures have been thoroughly studied. However, recently, a new source of error has been found: washing stains with "active oxygen" detergents abrogates presumptive and human hemoglobin tests for bloodstains (although visible). The aim of this investigation was to evaluate the ability of pure sodium percarbonate-main component of detergents-to abrogate presumptive and human hemoglobin tests. Then, a solution to this problem could be found. The results demonstrate that pure sodium percarbonate-itself-is able to abrogate all tests, as well as the different degrees to which each of them is affected by the product. Consequently, faced with a stain of bloody appearance, even the preliminary tests are negative; it is advisable to analyze the DNA. Otherwise, the opportunity of obtaining valuable information is lost.     

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.     

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.     

A blind trial evaluation of a crime scene methodology for deducing impact velocity and droplet size from circular bloodstains

In a previous study, mechanical engineering models were utilized to deduce impact velocity and droplet volume of circular bloodstains by measuring stain diameter and counting spines radiating from their outer edge. A blind trial study was subsequently undertaken to evaluate the accuracy of this technique, using an applied, crime scene methodology. Calculations from bloodstains produced on paper, drywall, and wood were used to derive surface-specific equations to predict 39 unknown mock crime scene bloodstains created over a range of impact velocities (2.2-5.7 m/sec) and droplet volumes (12-45 microL). Strong correlations were found between expected and observed results, with correlation coefficients ranging between 0.83 and 0.99. The 95% confidence limit associated with predictions of impact velocity and droplet volume was calculated for paper (0.28 m/sec, 1.7 microL), drywall (0.37 m/sec, 1.7 microL), and wood (0.65 m/sec, 5.2 microL).     

The use of error rates to predict and characterize the efficacy of presumptive field tests

From a forensic perspective, a presumptive test, one which indicates the presence or absence of a certain target material such as blood, is an invaluable tool. Among these tests, there are different specificities, sensitivities, and shelf lives. The accuracy of a test is an algebraic combination of the specificity and sensitivity of the test. Each test has limitations as given by its false positive and false negative rates. The aim of this study was to illustrate how the false positive and false negative rates are to be properly determined using a simulation study for the phenolphthalein test. New presumptive tests must be properly evaluated/validated through testing of commonly encountered household items and other potentially probative items usually found at crime scenes, however, the makeup of test sets must appropriately capture all error rates. In order to correctly use these results when the test is applied to an unknown sample recovered at a crime scene, the error rates cannot be applied directly to estimate whether or not the sample is actually the analyte of interest. In a validation study, the forensic scientist calculates the false positive rate as the p(Positive Reaction|Blood), whereas at the scene, the crime scene investigator wishes to determine the p(Blood|Positive Reaction). All crime scene investigators need to ensure that the conditional is not transposed when interpreting such results. Furthermore, this work provides a model for the assessment of a multiple test diagnostic system intended for investigators.     

The effect of luminol on presumptive tests and DNA analysis using the polymerase chain reaction.

This study was designed to test the following factors involved with processing luminol treated bloodstained evidence: 1) The reactivity of other presumptive chemical color tests, phenolphthalin (PT) and tetramethylbenzidine (TMB), following the application of the light emitting luminol presumptive test. 2) The effect of different cleanings of various bloody substrates on the luminol test. 3) The effect of different cleanings of various bloody substrates on the ability to obtain DNA suitable for PCR testing. 4) The ability to extract DNA from luminol treated bloodstained substrates using three extraction techniques. 5) The effect of spraying washed and unwashed bloodstains on various substrates with luminol on the ability to correctly type the DNA using PCR. Our findings indicated that luminol did not adversely effect the PCR testing and did not interfere with the PT and TMB presumptive tests for blood. It was determined that the substrate and the method of cleaning were the major factors affecting DNA yield and the ability to type the bloodstains using PCR based technologies.     

A Comparison of Four Presumptive Tests for the Detection of Blood on Dark Materials

Detection of blood on dark materials is difficult for crime scene examiners so presumptive tests are used to assist. This study compared the ability of luminol, leuko crystal violet, tetramethylbenzidine, and Combur Test®E to detect whole, diluted blood (1:100) and a key‐shaped blood transfer stain (1:10), on dark cotton sheeting, tea towel, socks, synthetic carpet, and car mats. Powdered bleach was used to evaluate specificity of the blood detection tests. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall misclassification rate (OMR) assessed the quality of the blood tests. Luminol was the preferred test for diluted blood having the highest sensitivity (79%–96%), NPV (66%–93%), and the lowest OMR (3%–15%). Luminol was also found to be most efficient with a testing time on 25 items of 2 h 50 min compared with up to 8 h. Overall, luminol was the most effective method, also providing information on bloodstain patterns.     

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.     

Influence of the luminol chemiluminescence reaction on the confirmatory tests for the detection and characterization of bloodstains in forensic analysis

Preliminary tests for the detection of stains at crime scenes aim to focus the police work making them more efficient in the combat of criminality. The application of the luminol chemiluminescence reaction (3-aminoftalhidrazida) in presumptive tests for the detection of bloodstains is known for more than 40 years in forensic science. This reaction is based on the emission of light through the chemical reaction of luminol mixed with hydrogen peroxide and a hydroxide in the presence of a catalytic molecule (iron from the hemoglobin) (Laux [1]).This work evaluates the luminol interference and its effect on subsequent serological and DNA testing. Samples prepared with blood and different concentrations of luminol solution containing luminol, peroxide of hydrogen and sodium carbonate, were analyzed. Additionally, samples of serial dilutions of standard DNA mixed with luminol solution were also analyzed. Although presumptive tests with luminol do not establish the characterization and identification of stains at crime scenes, preliminary results indicated that it is suitable for the detection of invisible bloodstains for forensic analysis, with few detrimental effects on the serological tests and subsequent DNA recovery and typing.     

Comparison of presumptive blood test kits including hexagon OBTI

Four presumptive blood tests, Hexagon OBTI, Hemastix(R), Leucomalachite green (LMG), and Kastle-Meyer (KM) were compared for their sensitivity in the identification of dried bloodstains. Stains of varying blood dilutions were subjected to each presumptive test and the results compared. The Hexagon OBTI buffer volume was also reduced to ascertain whether this increased the sensitivity of the kit. The study found that Hemastix(R) was the most sensitive test for trace blood detection. Only with the reduced buffer volume was the Hexagon OBTI kit as sensitive as the LMG and KM tests. However, the Hexagon OBTI kit has the advantage of being a primate specific blood detection kit. This study also investigated whether the OBTI buffer within the kit could be utilized for DNA profiling after presumptive testing. The results show that DNA profiles can be obtained from the Hexagon OBTI kit buffer directly.     

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.     

Evaluating the use of hypoxia sensitive markers for body fluid stain age prediction

To augment DNA profiling and body fluid identification techniques efforts are being made to increase the amount of information available from a crime scene stain, which includes efforts to identify externally visible characteristics through phenotypic analysis. A key question surrounding crime scene stains is the length of time between deposition of the stain and its subsequent recovery, in that is the stain recovered related to the incident in question or from a previously deposited stain number of weeks earlier? The inability to answer this fundamental question has a detrimental effect upon the successful completion of a criminal investigation. Once a body fluid leaves the body, the oxygen concentration in the environment changes; therefore, it may be that this change could cause a change in the expression of hypoxia-sensitive biomarkers. Here, a range of bloodstains, liquid saliva and liquid semen samples were collected at 0 days, 7 days, 14 days, 21 days and 28 days of degrading at room temperature (19–22 °C), before undergoing total RNA extraction and cDNA synthesis. Blood was recovered from filter paper with 3 mm², with saliva and semen being left in their tubes and swabbed at the appropriate times. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A (VEGFA) and Hypoxia-Inducible Factor 1 Alpha (HIF1A), with B-Actin (ACTB) as a reference gene. A range of linear and quadratic correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 4.2, 2.1, and 5 days for blood, saliva, and semen respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on samples up to four weeks old with a margin of error ranging from 2 days through to 5 days. While a sizeable potential time frame exists using this model; this represents a significant step towards the target of having an accurate stain age prediction model.     

“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.     

The collaborative study on typing group-specific component by eight forensic science laboratories

This report describes a collaborative study on typing group-specific component (GC), conducted between the Central Research and Support Establishment and the forensic science laboratories of England, Wales and Northern Ireland. A population study (n=114) was performed. Fifty blood donors were selected to provide a distribution, slightly biased from normal, in favour of the GC 1F-1F and GC 1F-1S phenotypes. A protocol was devised for preparing large bloodstains. The strongest GC bands were obtained from the edge of a stain after the blood had been treated with K+/EDTA. Each laboratory received a representative portion of the large bloodstains for GC typing. Five of the eight laboratories correctly grouped all the bloodstains. No errors directly attitributable to the system were recorded in over 800 tests, indicating that GC in bloodstains can be typed reliably using the combination of isoelectric focusing in ultrathin narrow pH interval gels followed by immunofixation and silver staining.     

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.     

D20S161和D8S384两个基因座在法医学中的应用

评估D2 0S16 1和D8S384两个基因座在法医学中的应用价值。用自制的D2 0S16 1和D8S384两个DNA分型试剂盒 ,对人血、人精液、人唾液、动物血、人血与动物血的混合检材和人血痕、人精液斑、人唾液斑、动物血痕、人血与动物血的混合斑痕检材 ,以及陈旧血痕检材进行检测分型 ,并用这两个基因座PCR引物序列与DNA数据库进行联网对比分析。自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血、人精液、人唾液、人血与动物血的混合检材分型 ,而动物血没有PCR产物 ;自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血痕、人精斑、人唾液斑和人血与动物血的混合斑痕检材正确分型 ,而动物血痕没有PCR产物 ;斑痕检材分型结果与对应体液检材分型结果无差异 ;5 0份陈旧血痕检材全部获得阳性分型结果。DNA数据库联网比较提示 ,D2 0S16 1和D8S384基因座引物除了能与各自的模板序列发生特异性扩增外 ,理论上不能与DNA数据库中 6 0 6 36 4种已知序列产生PCR产物。D2 0S16 1和D8S384两个基因座具有高度的种属特异性 ,抗污染能力强 ,不易受降解的影响 ,是解决法医现场生物检材个人识别和亲子鉴定的理想手段     

Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.     

Evaluation of a Visualization Assay for Blood on Forensic Evidence

In forensics, bloodstains on dark fabrics might be invisible for the naked eye. Although several visualization, presumptive, and confirmatory blood tests have been developed, all have one or more disadvantages, especially on DNA analysis. We report here the use of a visualization assay that can visually detect blood drops up to 1/20 dilution. In this assay, the fabric is placed between two wet filter papers and covered by glass surfaces on both sides. Pressure is applied on the glass surfaces in which bloodstains transfer onto the filter papers through capillary forces. Detected stains can be tested with other more sensitive presumptive blood tests performed on the filter paper. Even more, DNA analysis can be performed on the transferred bloodstains. The presented visualization assay is easy to perform, extremely cheap, requires little hands on time, and does not affect bloodstain pattern analysis.     

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.     

A study of the sensitivity and specificity of four presumptive tests for blood

The purpose of this work was to conduct a comparative study of the sensitivity and specificity of phenolphthalein, tetramethylbenzidine, leucomalachite green, and orthotolidine as presumptive tests for blood. The findings of this study indicate that the phenolphthalein and the leucomalachite green tests are the most specific and that the tetramethylbenzidine and orthotolidine tests are the most sensitive of the group. The author concludes that the phenolphthalein test is the best single test for evaluating suspected bloodstains.