Latent Bloodstains Research Articles

DNA degradation of bloodstains on cotton fabric caused by different washing procedures.

DNA degradation in biological material needs to be better understood. Bloodstains on washed clothing are disturbed by washing procedures, sometimes transferred to other fabrics, often with latent bloodstains and usually with significantly degraded DNA.The samples (cotton fabric with bloodstains) are divided into six main groups, depending on the washing method regarding water temperature (95, 60, and 30°C) and the detergent use. After completing the washing process, samples were stored for a certain period (1day to 6months) and subsequently analyzed. Analyses were performed using standard protocols and commercial kits to measure the remaining DNA quantity (concentration) and DNA degradation index in the processed samples.Our results revealed that the high washing temperature (60 and 95°C) and the application of detergent have a synergic action on DNA degradation, while at 30°C this effect is absent. Furthermore, the effect of detergent on accelerated DNA degradation is observed about a month after the washing. This delayed effect of detergent has no explanation in current literature data.To obtain optimal results from the bloodstains, we recommended that the period from the crime event and attempted cleaning by a perpetrator to the laboratory analysis should be less than 1month.

Latent bloodstain detection using a selective turn-on NIR fluorescence dye responsive to serum albumin.

Bloodstain detection can provide crucial information and evidence at a crime scene; however, the ability to selectively detect bloodstains in a non-destructive manner with high sensitivity and low background is limited. This study reports a fluorescent dye (sulfonate indolizine squaraine, SO3SQ) for bloodstain visualization under near-infrared (NIR) irradiation. While the dye itself is minimally fluorescent in aqueous solution, it exhibits a "turn-on" mechanism upon binding with human serum albumin (HSA) as the fluorescence intensity increases over 160 times with strong absorption and emission at 693 nm and 758 nm, respectively. Bloodstains can be visualized on a surface even after being diluted 1000 times, and washed latent bloodstains can be detected with high sensitivity. Further, the turn-on fluorescent emission lasts for a minimum of seven days, allowing adequate time for detection. This study also indicates that the SO3SQ can sensitively detect bloodstain after the bloodstain aged for one week. Furthermore, the detection of latent blood fingerprint patterns from colorful backgrounds is demonstrated using this non-destructive method. The selective turn-on fluorescent dye with NIR excitation and emission is highly suitable in forensic science for bloodstain visualization.

Evaluation of infrared photography for latent bloodstain visualization and the influence of time

The localization of latent blood traces at crime scenes is generally performed using fluorescent stains although infrared light has previously been recognized as an effective localization test for bloodstains as it is a non-destructive and non-contact technique. The goal of this study was to evaluate infrared photography for the detection of latent bloodstains on a large number of objects with different compositions frequently encountered at crime scenes. In this study we show that infrared light photography was able to detect bloodstains deposited on 71.7% of materials while bloodstains on 29.2% of materials could only be detected using infrared photography and not through visual photography. Bloodstain could be detected on most fabrics composed of 100% polyester, 100% cotton and 100% wool or a combination of these fibers with other types of fiber such as nylon or viscose. For other materials such as leather, tiles, wood, bricks, parquet, infrared did not improve the visibility of the bloodstains. Finally, the influence of the time of bloodstain deposition was tested over the period of 1 week and 1 month and did not reveal major differences compared to stains after 24 h drying time suggesting that time has little influence on the ability of infrared light and visual light to detect latent bloodstains.

Occult bloodstains detection in crime scene analysis

Blood detection in forensic investigation is the milestone in crime scene analysis. Locating bloodstains on dark clothing are often difficult and it is well known the discrepancy about selection of the most suitable method to locate and identify blood stains, depending on the presence of different contaminants and the occurrence of false positives or negatives of human’s blood.The main purpose of this paper was to compare different orientation tests for blood detection on different materials, presenting with contaminated samples and treatments. Human blood was tested in car mat, dark tiles, unlacquered wood surface and sneakers with recent and old stains. Samples were prepared with blood, paint, bleach, dish soap multi-use cleaner, juice, fruit and milk using different combinations and protocols.In the case of forensic lights, and depending on the wavelength and filter used, latent stains were not detected and some false negatives and false positives appeared in different contaminants and treatments. In addition, bleach resulted the main contaminant, altering most tests.In conclusion, our results confirmed that luminol is the most effective test for detecting fresh and latent blood stains, because no type of contamination interfered on its reliability.

Degrees of contrast: Detection of latent bloodstains on fabric using an alternate light source (ALS) and the effects of washing.

Blood is often a piece of evidence of violent crimes and will often be on the perpetrator's clothing. If the perpetrator is wearing dark clothing, it can be easily identified by chemical means such as Bluestar®, but this can destroy the pattern, which may be evidentiary itself. This study explores the use of alternate light source (ALS) to photograph bloodstains on dark and/or patterned fabrics to provide an alternate, noninvasive tool before the use of chemical detection techniques. Sixty-nine (69) unwashed fabrics, of various dark colored and dark patterns, were photographed in monochrome under ambient light and subsequently with and without a filter under ultraviolet (UV), violet, blue, green, and infrared light. This study used ImageJ to measure the contrast between the bloodstain and the fabric and thus the effectiveness of each wavelength. Each fabric was washed, photographed, and analyzed five times or until the bloodstain was no longer visible under ALS. Results indicated photography with ALS was a viable method for blood detection on fabrics and should be used prior to chemical means. Further, infrared, followed by violet light with no filter, was the most effective light source for viewing bloodstains on dark fabrics without the use of chemicals. However, these wavelengths were not effective on military uniforms. This study also described one effect fabric manufacturer chemical treatments have on bloodstains and the effect of washing fabrics with bloodstains.

MRNA profiling in casework analyses

Our work involved the application of mRNA profiling to three bloodstains previously processed by the laboratory, in which routinely methods (immunochromatography) used for specific blood identification showed negative results. Analyses were accomplished on a sweater worn by the suspect. No bloodstains were found during the routine inspection. In order to identify possible latent bloodstains, luminol was applied. After the reagent vaporization, three distinct areas of luminescence appeared. In order to confirm the presence of the human hemoglobin, luminescent surfaces were collected using 4N6FLOQSwabs ® and tested using HemDirect Hemoglobin test (SERATEC ® ). All samples analysed showed negative results. Due to the merging blood-specific markers mRNA profiling, the areas previously identified were sampled newly and tested using the three blood specific markers HBB, ALAS2 and CD93, together with two housekeeping genes represented by ACTB and 18S-rRNA. All samples showed positive results for all three blood specific mRNA markers.

A comparison of ABAcard® Hematrace® and RSIDTM-Blood tests on dried, diluted bloodstains treated with leucocrystal violet or luminol

ABSTRACT Presumptive tests for blood, such as luminol and leucocrystal violet (LCV), are often used by forensic officers when screening for latent (non-visible) or diluted bloodstains at crime scenes. Where positive reactions are observed, a confirmatory test for the presence of blood may be implemented. This study aimed to compare the efficacy of two such confirmatory tests (ABAcard® Hematrace® and RSIDTM-Blood) on dried, diluted bloodstains that were enhanced using either the Grodsky luminol formulation or LCV. Eighteen replicates per dilution (1:10, 1:100), enhancement, and confirmatory test were performed (144 bloodstains). The RSIDTM-Blood test produced false negative results for all luminol-enhanced bloodstains, regardless of dilution. This test performed slightly better for bloodstains enhanced with LCV, returning approximately 50% positive results. In contrast, the ABAcard® Hematrace® test performed well, returning positive detections for all luminol-treated bloodstains, and all but two LCV-enhanced stains (both 1:100 dilution). Significant differences were observed between the test results and suggested a potential inhibitory effect on the RSIDTM-Blood test from the Grodsky luminol formulation and, to a lesser extent, LCV. This research has demonstrated that the RSIDTM-Blood test is not a reliable confirmatory test faint or latent bloodstains enhanced with luminol or LCV.

Detection of latent bloodstains at fire scenes using reflected infrared photography

Bloodstain evidence is an element of crime scene investigation often found at scenes involving violence. Setting fire to the scene is a method sometimes used by offenders of crime in an attempt to conceal evidence. Fire often produces thick soot as a by-product of the combustion and has the potential to cover bloodstain patterns rendering them latent. There is limited published material offering a method of detecting bloodstains hidden beneath dense soot deposits caused by fire. This project employed a modified digital single-lens reflex (SLR) camera to investigate the application of reflected infrared photography to detect latent bloodstain evidence beneath varying deposited overlaying soot densities. The potential of this technique was examined by photographing blood samples beneath soot from a scaled fire simulation. A qualitative evaluation was completed by comparing images taken of a series of samples using both reflected infrared and standard visible light photography and corroborated with quantitative image analysis to support the findings. Results indicate that infrared photography can reveal latent bloodstains beneath a dense layer of soot in excess of ρ2.3 (550nm) density with substantial clarity. The success of this technique is dependent on specific optical and specimen parameters. These parameters include (i) the reflective properties of the background surface, (ii) the spectral absorption properties of blood and (iii) the ability of infrared wavelengths to transmit through the soot layer. Reflected infrared photography may provide crime scene examiners with a specialised field recording method that is easily executed and non-destructive to assist in visualising and locating latent bloodstain patterns beneath dense layers of soot.

Modular-Based Integrated Microsystem with Multiple Sample Preparation Modules for Automated Forensic DNA Typing from Reference to Challenging Samples.

The realization of an automated short tandem repeat (STR) analysis for forensic investigations is facing a unique challenge, that is DNA evidence with wide disparities in sample types, quality, and quantity. We developed a fully integrated microsystem in a modular-based architecture to accept and process various forensic samples in a "sample-in-answer-out" manner for forensic STR analysis. Two sample preparation modules (SPMs), the direct and the extraction SPM, were designed to be easily assembled with a capillary array electrophoresis (CAE) chip using a chip cartridge to efficiently achieve an adequate performance to different samples at a low cost. The direct SPM processed buccal swabs to produce STR profiles without DNA extraction in about 2 h. The extraction SPM analyzed more challenging blood samples based on chitosan-modified quartz filter paper for DNA extraction. This newly developed quartz filter provided a 90% DNA extraction efficiency and the "in situ" PCR capability, which enabled DNA extraction and PCR performed within a single chamber with all the DNA concentrated in the filter. We demonstrated that minute amounts of blood (0.25 μL), highly diluted blood (0.5 μL blood in 1 mL buffer), and latent bloodstains (5-μL bloodstain on cloth washed with detergent) can be automatically analyzed using our microsystem, reliably producing full STR profiles with a 100% calling of all the alleles. This modular-based microsystem with the capability of analyzing a wide range of samples should be able to play an increasing role in both urgent situations and routine forensic investigations, dramatically extending the applications and utility of automated DNA typing.

Robust Serum Albumin-Responsive AIEgen Enables Latent Bloodstain Visualization in High Resolution and Reliability for Crime Scene Investigation.

Bloodstains provide admissible information for crime scene investigators. The ability to resolve latent bloodstains that are commonly found in real scenarios is therefore pivotal to public security. Here, we report a facile approach for invisible bloodstain visualization based on the click reaction between serum albumin and tetraphenylethene maleimide (TPE-MI), an aggregation-induced emission luminogen (AIEgen). Compared to the widely adopted methods based on the harsh catalytic oxidation activity of hemoglobin, this working principle benefits from the specificity of the mild catalyst-free thiol-ene click reaction that improves the reliability and resolution. In addition, the mild conditions preserve DNA information and bloodstain patterns, and the excellent photophysical properties of the AIEgen afford high sensitivity and stability (>1 yr). Such an excellent performance cannot be achieved by conventional AIEgens and aggregation-caused quenching luminogens with similar structures. TPE-MI outperforms the benchmark luminol-based technique in visualizing latent bloodstains as showcased in two mock crime scenes: spattered blood track and transfer blood fingerprint. This disclosed method is an advancement in forensic science that could inspire future development of technology for bloodstain visualization.

A quantitative method for determining a representative detection limit of the forensic luminol test for latent bloodstains

The luminol test has been used for over 60 years by forensic investigators for presumptive identification of blood and visualization of blood splatter patterns. Multiple studies have estimated the limit of detection (LD) for bloodstains when luminol is employed, with results ranging from 100× to 5,000,000× dilute. However, these studies typically have not identified and controlled important experimental variables which may affect the luminol LD for bloodstains. Without control of experimental parameters in the laboratory, variables which affect the potential of presumptive bloodstain test methods remain largely unknown, and comparisons required to establish new, more powerful detection methods are simply impossible. We have developed a quantitative method to determine the relationship between the amount of blood present and its reaction with luminol by measuring, under controlled conditions, the resulting chemiluminescent intensity with a video camera, combined with processing of the digital intensity data. The method resulted in an estimated LD for bloodstains on cotton fabric at ∼200,000× diluted blood with a specific luminol formulation. Although luminol is the focus of this study, the experimental protocol used could be modified to study effects of variables using other blood detection reagents.

Artemisinin-Luminol Chemiluminescence for Forensic Bloodstain Detection Using a Smart Phone as a Detector.

Forensic luminol chemiluminescence test is one of the most sensitive and popular methods for the determination of latent bloodstains. It mainly uses hydrogen peroxide or sodium perborate as coreactants. The easy decomposition of hydrogen peroxide and sodium perborate in the presence of many ions significantly affects the selectivity. Artemisinin is a natural peroxide that is quite stable in the presence of common ions. In the present study, artemisinin has been exploited for the forensic bloodstain chemiluminescence detection for the first time. Using smart phone as cost-effective portable detector, the visual detection of bloodstains has been achieved with a dilution factor of blood up to 100 000. Moreover, this system shows excellent selectivity against many common species. It can well differentiate bloodstains from other stains, such as coffee, brown sugar, and black tea. Both favorable sensitivity and selectivity makes the present method promising in forensic detection.

Duration and intensity of chemiluminescence of different luminol kits

The use of chemiluminescent bloodstain detection reagents to investigate crime scenes is well established, especially when detection of latent bloodstains is needed. The application instructions of commonly used luminol products recommend prompt usage of freshly mixed chemical agents for optimal visualization of highly visible and long-lasting chemiluminescent bloodstains; however, scene processing efforts can delay bloodstain examinations and immediate usage. This study was conducted with the objective of comparing the applicability of two different luminol products (Bluestar® and Lumiscene Ultra) on invisible, standardized bloodstains over a period of 7 days. The mixed luminol reagents were constantly stored at 4 °C. For the study freshly drawn blood diluted in water (1:50) were dropped from a height of 30 cm onto tiled surfaces, producing mean drop diameters of 10–12 mm. From these blood drops 10 were randomly chosen and a fine mist of luminol reagent was applied using a regular spraying distance of 100 cm. Following photo documentation of the spots, measurements of both intensity and duration of luminescence were performed using Adobe Photoshop®. Up to day 5 following application, a constant and expected bright light blue pattern of chemiluminescence could be observed despite already decreasing digitally based luminescence values. Lumiscene Ultra revealed a slightly longer duration of luminescence of unchanged intensity during the first 3 days, whereas Bluestar® resulted in earlier decrease in values of light intensity after 2 days but with longer overall duration of chemiluminescence.

Ascorbic acid and vitamin C-containing beverages delay the leucomalachite green reaction to detect latent bloodstains.

The leucomalachite green (LMG) test is one of catalytic tests for the detection of latent bloodstains and generally used in forensic field because of convenience and cost/time-effectiveness. However, contamination of latent bloodstains at crime scenes can interfere with the LMG reaction, resulting in false-negative or false-positive decisions. Herein, we examined if ascorbic acid and vitamin C (l-ascorbic acid or ascorbate)-containing beverages affect the LMG reaction. Ascorbic acid showed the inhibitory activities on the LMG reaction in a dose-dependent manner. Similarly, vitamin C-containing beverages also inhibited the LMG reaction and the inhibitory effects were proportional to the concentrations of vitamin C in beverages. It was also identified that as incubation time after adding LMG reagent to the mixtures of blood and ascorbic acid or beverages was increased, the inhibitory effects of ascorbic acid vitamin C-containing beverages on LMG test were disappeared. These results suggest that the LMG reaction is delayed but not stopped by ascorbic acid and vitamin C-containing beverages. Neither incubation at room temperature around 20-25°C nor the addition of acetic acid affects the inhibitory activity of ascorbic acid on LMG reaction. We also showed that ascorbic acid does not affect DNA stability, allowing us to obtain full short tandem repeat (STR) profiles through amplification of DNA using commercial STR kits. In conclusion, ascorbic acid and vitamin C-containing beverages delayed the LMG reaction, suggesting that it should be considered that negative results of LMG test could be false negative due to contamination of bloodstains with inhibitory factors on LMG test.

The non-contact detection and identification of blood stained fingerprints using visible wavelength reflectance hyperspectral imaging: Part 1

Blood is one of the most commonly encountered types of biological evidence found at scenes of violent crime and one of the most commonly observed fingerprint contaminants. Current visualisation methods rely on presumptive tests or chemical enhancement methods. Although these can successfully visualise ridge detail, they are destructive, do not confirm the presence of blood and can have a negative impact on DNA sampling.A novel application of visible wavelength reflectance hyperspectral imaging (HSI) has been used for the detection and positive identification of blood stained fingerprints in a non-contact and non-destructive manner on white ceramic tiles. The identification of blood was based on the unique visible absorption spectrum of haemoglobin between 400 and 500nm.HSI has been used to successfully visualise ridge detail in blood stained fingerprints to the ninth depletion. Ridge detail was still detectable with diluted blood to 20-fold dilutions. Latent blood stains were detectable to 15,000-fold dilutions. Ridge detail was detectable for fingerprints up to 6months old. HSI was also able to conclusively distinguish blood stained fingerprints from fingerprints in six paints and eleven other red/brown media with zero false positives.

Black and green tea – How to make a perfect crime

The antioxidant properties of the black and green tea are well known. The latent bloodstains are detectable by luminol. The bloodstains also can be cover up by drinks and foods containing the antioxidants; thus their presence can cause a decrease of the luminol light emission (false-negative results). The aim of this study was to quantify the light emission decrease of the chemiluminescent mixture prepared according to Weber (containing NaOH) and the chemiluminescent mixture of pH 7.4 (for the determination of the total antioxidant capacity) for the open air-dried sample. The black and green teas and white wine were used as the antioxidant's samples (high and low total antioxidant capacity). The significant decrease of the luminol chemiluminescent emission caused by the presence of the black and green teas (and comparable for both of them) was observed in comparison with the presence of white wine.

An evaluation of luminol formulations and their effect on DNA profiling

Luminol is a presumptive test reagent used for the location of latent bloodstains. Various formulations are used by different forensic practitioners and commercial products are also widely available. There is little concurrence between authors with regards to the sensitivity limits of luminol which can vary significantly depending upon the substrate. We evaluated the sensitivity and stability of five different luminol formulations on a range of blood dilutions. All formulations showed an overall decrease in performance over 24h though the effect was more gradual on a non-porous surface compared to porous. We found that BlueStar® Magnum showed the greatest sensitivity compared to other formulations and detected 50μl of 1/100,000 blood dilutions on both porous and non-porous surfaces. Two formulations of luminol were selected based on the result of the sensitivity and stability study and were assessed for their impact on the DNA profiling process. There was a statistically significant improvement in DNA profile peak area from luminol-treated samples when compared to control samples of neat blood stains. However, at the weaker blood dilution of 1/1,000, the difference between control and luminol-treated samples was dependent on the substrate type with porous (fabric) samples showing a significant difference and non-porous (tile) swabbed samples requiring further work to conclusively ascertain the effect.

Preliminary Observations on the Ability of Hyperspectral Imaging to Provide Detection and Visualization of Bloodstain Patterns on Black Fabrics

The analysis of bloodstain patterns can assist investigators in understanding the circumstances surrounding a violent crime. Bloodstains are routinely subjected to pattern analysis, which is inherently dependent upon the ability of the examiner to locate and visualize bloodstain patterns on items of evidence. Often, the ability to properly visualize bloodstain patterns is challenging, especially when the stain patterns occur on dark and/or patterned substrates. In this study, preliminary research was performed to better understand how near-infrared reflectance hyperspectral imaging (HSI) could be used to observe bloodstain patterns on commonly encountered black fabrics. The ability of HSI to visualize latent bloodstains on several commonly encountered substrates is demonstrated. The images acquired through HSI are of sufficient quality to allow for differentiation between stains produced from an impact mechanism or a transfer mechanism. This study also serves as a proof of concept in the differentiation of multiple staining materials. Because of its ability to generate spectral data, the data provide a preliminary separation of stains where more than one type of stain existed.

Evaluation of the catalytic decomposition of H2O2 through use of organo-metallic complexes – A potential link to the luminol presumptive blood test

Forensic scientists use several presumptive tests to detect latent blood stains at crime scenes; one of the most recognizable being the luminol reagent. Luminol, under basic conditions, reacts with an oxidizing species which, with the help of a transition metal catalyst facilitates a luminescent response. The typical oxidizing species used in the luminol reaction is hydrogen peroxide (H(2)O(2)). While the luminol reaction has been studied since its inception, the mechanistic pathway is still an area of great debate. Previous work suggests that the luminol reaction with latent blood stains possesses a correlation to the Fenton-Decomposition reaction mechanism, which decomposes H(2)O(2) into the strongly oxidizing hydroxyl radical (*OH) species. This work seeks to understand the luminol reaction on a mechanistic level and to determine if a synergy exists between the chemiluminescence observed in the reaction and the production of the hydroxyl radical via Fenton-like processes. Results indicate that organo-metallic complexes produce hydroxyl radicals at different rates and different concentrations. These findings appear to be related to structural differences in the organo-metallic complex, which conform to the 18 electron rule or are one electron rich/deficient. Furthermore, the production of *OH is controlled by the chemical environment which governs complex stability at high pH conditions, reflective of the luminol process. Model hemoglobin systems reveal a strong correlation between the rate of *OH production via the Fenton-like pathway and maximum chemiluminescent intensity.

Investigation of Physical Effects of Acid Yellow 7® Enhancement on Dark and Non-Porous Surfaces in Impact Pattern Area of Origin Estimation

Impact patterns that are generated on dark surfaces can become camouflaged creating difficulties with conventional photographic methods that are used when estimating the area of origin. Chemical enhancement is an alternative technique that can be used to visualize these latent bloodstains. The application of Acid Yellow 7 to already fixed bloodstains provides a robust enhancement protocol which yields minimal stain distortion. Image J software was utilized to count stains, resulting in an approximate 5% increase in small stain detection post-enhancement. Area analyses also performed with Image J demonstrates that the average area of a single stain increases by a factor of approximately 1.24 post-enhancement. When using the BackTrack™ suite of programs this enlargement does not significantly affect the calculated impact angle of an enhanced bloodstain when compared to that of its original deposition (P= 0.585). As a result, the area of origin can be estimated within the accepted error of ± 7 cm from the known blood source.