Does Sunlight Affect the Quality for Purposes of DNA Analysis of Blood Stain Evidence Collected from Different Surfaces?

The aim of this study was to investigate the effect of sunlight on the degradation of DNA samples taken from blood stains from different types of surfaces. A blood sample obtained from a single male donor was placed on seven different surfaces (galvanized sheet, iron rod, newspaper, white printer paper, glass, soil, and ceramic panel). Samples were kept, during a 4-week summer period, in a room, but next to an open window. Every 7 days, 1 mm2 of blood sample was collected from each substrate and stored in labeled tube for later analysis. DNA was extracted with the Chelex method, amplified using AmpFISTRTM MinifilerTM Plus Amplification Kit, and quantified using a QuantifilerTM Human DNA Quantification kit. After 7 days of sun exposure, the highest DNA concentration was determined to be from the sample from a galvanized sheet stain, followed by, in order of decreasing concentration, the ceramic panel, glass, newspaper, iron rod, and white printer paper surface. As expected, the DNA concentration from all samples decreased as the sunlight exposure time progressed. The results obtained after the amplification in the MiniFilerTM system were in correlation with the DNA concentrations measured by the qPCR method for all samples, except for the glass, soil, and white printer paper samples. The obtained data show that DNA degradation is correlated to the length of sunlight exposure and to the type of surface the samples are collected from. A negative qPCR result does not mean negative PCR amplification in the STR system; therefore, both methods should be applied when analyzing forensic samples collected from trace evidence.

Correction of Substrate Spectral Distortion in Hyper-Spectral Imaging by Neural Network for Blood Stain Characterization.

In the recent past, hyper-spectral imaging has found widespread application in forensic science, performing both geometric characterization of biological traces and trace classification by exploiting their spectral emission. Methods proposed in the literature for blood stain analysis have been shown to be effectively limited to collaborative surfaces. This proves to be restrictive in real-case scenarios. The problem of the substrate material and color is then still an open issue for blood stain analysis. This paper presents a novel method for blood spectra correction when contaminated by the influence of the substrate, exploiting a neural network-based approach. Blood stains hyper-spectral images deposited on 12 different substrates for 12 days at regular intervals were acquired via a hyper-spectral camera. The data collected were used to train and test the developed neural network model. Starting from the spectra of a blood stain deposited in a generic substrate, the algorithm at first recognizes whether it is blood or not, then allows to obtain the spectra that the same blood stain, at the same time, would have on a reference white substrate with a mean absolute percentage error of 1.11%. Uncertainty analysis has also been performed by comparing the ground truth reflectance spectra with the predicted ones by the neural model.

Forensic applications of hyperspectral imaging technique: a narrative review.

Hyperspectral imaging (HSI) collects and processes information from the entire electromagnetic spectrum to obtain the spectrum of each pixel in the image of a scene, with the aim of finding objects and identifying materials. It is a non-contact, non-destructive technology that can be used without modifying or altering the analysed target. Forensic analysis and crime scene investigations are two of the most investigated fields of application, being able to detect and analyse many types of evidence.In this paper we analysed the most commonly reported forensic science applications.The literature indicates that the fields in which HSI appears most promising are the analysis of blood traces, document forgery, gunshot residues and the identification of fingerprints.

Hierarchical classification models and Handheld NIR spectrometer to human blood stains identification on different floor tiles.

One of the most important types of evidence in certain criminal investigations is traces of human blood. For a detailed investigation, blood samples must be identified and collected at the crime scene. The present study aimed to evaluate the potential of the identification of human blood in stains deposited on different types of floor tiles (five types of ceramics and four types of porcelain tiles) using a portable NIR instrument. Hierarchical models were developed by combining multivariate analysis techniques capable of identifying traces of human blood (HB), animal blood (AB) and common false positives (CFP). The spectra of the dried stains were obtained using a portable MicroNIR spectrometer (Viavi). The hierarchical models used two decision rules, the first to separate CFP and the second to discriminate HB from AB. The first decision rule, used to separate the CFP, was based on the Q-Residual criterion considering a PCA model. For the second rule, used to discriminate HB and AB, the Q-Residual criterion were tested as obtained from a PCA model, a One-Class SIMCA model, and a PLS-DA model. The best results of sensitivity and specificity, both equal to 100%, were obtained when a PLS-DA model was employed as the second decision rule. The hierarchical classification models built for these same training sets using a PCA or SIMCA model also obtained excellent sensitivity results for HB classification, with values above 94% and 78% of specificity. No CFP samples were misclassified. Hierarchical models represent a significant advance as a methodology for the identification of human blood stains at crime scenes.

[The study of blood stains at the Department of Forensic Medicine of the Sechenov University over the period of its existence]. / Izuchenie sledov krovi na kafedre sudebnoi meditsiny Sechenovskogo universiteta za period ee sushchestvovaniya.

The historical analysis and the results of research activities of the Department of Forensic Medicine of the Sechenov University on the study of blood stains as important material evidence in forensic medicine and forensic science are presented. The personal contribution to the solution of this problem of outstanding forensic doctors whose life and work was associated with the department was noted.

Development of Integrated Device of Trace Bloodstains Imaging and Age Analysis.

ABSTRACT: Objective To develop a device of trace bloodstains imaging and age analysis, so as to provide a non-destructive, simple and objective method for age estimation of bloodstains at the crime scene. Methods Based on the principle of digital imaging and color pattern analysis, the mobile terminal of the device was used to collect images of bloodstains of different ages. The time-dependent pattern of 6 parameters （R, G, B, C, Y, M） reflecting the changes of color of images of different ages was obtained by computer image analysis. A multiparameter comprehensive inference equation of bloodstains age was established and embedded into the device software to realize the intelligent inference of the bloodstains age. Then the capability and reliability of the device was verified. Results This integrated device of bloodstains imaging and age analysis could quickly collect bloodstains at the crime scene and automatically analyze and infer the age of bloodstains combined with related intelligence software. In the blind test, the detection accuracy of this device was 95% in both natural light airtight group and dark airtight group, and 80% in the natural light ventilation group. Conclusion The integrated device of trace bloodstains imaging and age analysis can be used in a simple manner, which provides a new objective method for bloodstains age estimation.

Detectability of bloodstains after machine washing.

Bloodstains on textiles can provide useful information for the forensic reconstruction of a crime. Surprisingly, little is known about the applicability of bloodstain traces after a textile was machine washed. In this study, we investigated the effect of machine washing on bloodstains on both cotton and polyester cloths. The influence of the washing detergent, the type of washing machine, the washing temperature, and the duration of drying of the bloodstain prior to washing as well as the drying temperature was investigated. Additionally, the molecular analyses of a subsample of the experiments were conducted. We found that although the primary morphology of the traces is often blurred, the presence of blood on the textiles can still be detected in many cases. Blood can also be transmitted to previously blood-free textiles during the washing process, leading to a positive Luminol or Combur® reaction of these samples. When traces of blood can be detected via the Luminol reaction, a molecular identification of the blood donor was successful in 28% of the cases.

Development of Integrated Device of Trace Bloodstains Imaging and Age Analysis / 法医学杂志

Objective To develop a device of trace bloodstains imaging and age analysis, so as to provide a non-destructive, simple and objective method for age estimation of bloodstains at the crime scene. Methods Based on the principle of digital imaging and color pattern analysis, the mobile terminal of the device was used to collect images of bloodstains of different ages. The time-dependent pattern of 6 parameters （R, G, B, C, Y, M） reflecting the changes of color of images of different ages was obtained by computer image analysis. A multiparameter comprehensive inference equation of bloodstains age was established and embedded into the device software to realize the intelligent inference of the bloodstains age. Then the capability and reliability of the device was verified. Results This integrated device of bloodstains imaging and age analysis could quickly collect bloodstains at the crime scene and automatically analyze and infer the age of bloodstains combined with related intelligence software. In the blind test, the detection accuracy of this device was 95% in both natural light airtight group and dark airtight group, and 80% in the natural light ventilation group. Conclusion The integrated device of trace bloodstains imaging and age analysis can be used in a simple manner, which provides a new objective method for bloodstains age estimation.

[The analysis of the results of the medical criminalistics expertises of the blood stains performed at the Bureau of Forensic Medical Expertise of the Moscow Health Department during the period from 2011 till 2015]. / Analiz mediko-kriminalisticheskikh ékspertiz sledov krovi, vypolnennykh v Biuro sudebno-meditsinskoi ékspertizy Departamenta zdravookhraneniia Moskvy za period 2011-2015 gg.

The objective of the present study was the analysis of the results of the medical criminalistics expertises of the blood stains with reference to their different characteristics including the occurrence across the years, variability, the forms and parameters of the traumatizing impacts, the properties of the objects with which the injuries were inflicted, etc. The majority of the data necessary for the analysis were extracted from the archive materials of the Medical and Criminalistic Department of the Bureau of Forensic Medical Expertise of the Moscow Health Department for the period from 2011 till 2015. A total of 5749 expertises performed during this time included 107 analyses of blood stains, 98 trace-evidence and 9 situational analyses. The specific features of such expertises include a large number of objects carrying blood stains available for the analysis. In the majority of the cases, such objects are stained with blood of several types differing in the mechanisms of their formation. 3D-scanning of the scene of the event with the subsequent three-dimensional computer-assisted simulation of the actions of each participant of the accident is considered to be the most promising method for the medical criminalistic expertise of the blood stains. The application of this approach is believed to make the work of the forensic medical experts significantly easier.

[Effect of Benzidine Test on DNA Analysis of Bloodstain].

OBJECTIVES: To explore the effect of benzidine test and related reagents on DNA analysis of bloodstain. METHODS: A total of 970 bloodstain filter paper samples with 1 µL venous blood were collected, and 10 of them acted as control samples. After benzidine test and related reagent processing, DNA of 960 samples was extracted by Chelex-100 and silica bead methods and then multiplex amplified by AmpFℓSTR™ Identifiler™ Plus PCR kits. The results of STR typing were compared between different groups. RESULTS: DNA were extracted immediately after benzidine test. Totally STR loci （3.80±1.34） were detected by silica bead method, while no STR loci were obtained by Chelex-100 method. Thirteen samples （21.7%） with whole STR typing results were obtained by drying after benzidine test, and the STR locus number （12.90±1.49） which obtained by silica bead method was much higher than by Chelex-100 method （4.70±1.96） （P<0.05）. When DNA was extracted immediately after the addition of glacial acetic acid, the STR locus number was （9.40±2.09） by silica bead method, but no STR typing result was obtained by Chelex-100 method. All 15 STR loci could be obtained by only adding glacial acetic acid after drying and only adding tetramethylbenzidine alcoholization liquid or 3% hydrogen peroxide liquid. CONCLUSIONS: Benzidine test has significant influence on DNA analysis of bloodstain. The Chelex-100 method is not suitable for the DNA extraction of bloodstain after benzidine test. Drying after benzidine test and silica bead methods can effectively enhance the STR locus number of bloodstain.

[The specific features of the blood stains depending on their volume]. / Osobennosti sledov kapel' krovi pri ikh razlichnom ob''eme.

The objective of the present study was to determine the minimal and maximum volume of blood droplets freely falling from various droplet-forming surfaces and to characterize the specific morphological features of the resulting stains. The experiments were designed so that the blood droplets were allowed to fall down from the height of 10 cm to 1.5 m. The drop volume varied from 5 mcl to 134 mcl depending on the shape of the objects and the area of the droplet-forming surfaces. The size of the blood stains ranged from 0.4 to 2.4 cm. The stains of a smaller size were regarded as splashes while a stain resulting from the fall of a droplet having a volume greater than 200 mcl was considered to be a result of merging of several droplets and was termed 'blood volume'. The morphological features of the blood stains of different volume are described.

[The peculiar morphological features of the blood stains on the snow over of different density]. / Morfologicheskie osobennosti sledov kapel' krovi na snezhnom pokrove raznoi plotnosti.

The objective of the present study was to elucidate the peculiar morphological features of the blood stains on the snow over of different density. We have undertaken the modeling and investigation of the blood stains on the loose snow cover (ρ=0.35±0.03 g/cm3) and at the areas of snow compacted by a motor-vehicle wheel (ρ=0.96±0.03 g/cm3) with the use of the visual, metric, descriptive, and statistical methods. The analysis of the blood stain patterns on the loose snow showed that a blood drop tends to form a depressed cylindrical canal at the bottom of which an irregularly shaped spherical element can be seen; it is composed of small snow crystals soaked with blood. The upper end of the canal has the fine-scalloped edge tending to shed unevenly. The morphological picture of the blood stains at the compacted snow cover is similar to that on the hard wettable rough surfaces; they have the oval shape and the unevenly serrated edge with the well apparent signs of sputtering.at the periphery.

[The peculiar morphological features of the imprints of straight and wavy head hair dirtied with blood]. / Osobennosti otpechatkov priamykh i volnistykh volos golovy, ispachkannykh krov'iu.

The objective of the present study was to elucidate the specific morphological features of the imprints of blood-soaked straight and wavy head hair. The contact imprints of straight and wavy head hair dirtied with blood were obtained experimentally. The imprints of straight hair were shown to exhibit the elements in the form of the rectilinear and bow-shaped slightly bent stripes. The imprints of wavy hair were shaped as the arches, waves, circles, and a large number of various small elements, such as dashes and commas.

Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2.

Proteolytic bacteria isolated from municipal solid wastes (MSW) were identified as Serratia marcescens A3 and Pseudomonas putida A2 based on 16S rDNA sequencing. Protease produced through fermentation of organic MSW by these bacteria under some optimized physicochemical parameters was partially purified and characterized. The estimated molecular mass of the partially purified protease from S. marcescens and P. putida was approximately 25 and 38 kDa, respectively. Protease from both sources showed low Km 0.3 and 0.5 mg ml-1 and high Vmax 333 and 500 µmole min-1 at 40 °C, and thermodynamics analysis suggested formation of ordered enzyme-substrate (E-S) complexes. The activation energy (Ea) and temperature quotient (Q10) of protease from S. marcescens and P. putida were 16.2 and 19.9 kJ/mol, and 1.4 and 1.3 at temperature range from 20 to 40 °C, respectively. Protease of the both bacterial isolates was serine and cysteine type. The protease retained approximately 97% of activity in the presence of sodium dodecyl sulphate. It was observed that the purified protease of S. marcescens could remove blood stains from white cotton cloth and degrade chicken flesh remarkably. Our study revealed that organic MSW can be used as raw materials for bacterial protease production and the protease produced by S. marcescens A3 might be potential for applications.

Identification of female-specific blood stains using a 17ß-estradiol-targeted aptamer-based sensor.

Blood stain evidence obtained from a violent crime scene provides decisive clues that can enable a case to be solved through forensic analyses such as genetic identification. However, collected samples usually contain a mixture of biological material from different sources, making genetic identification difficult. To address this issue, we developed an activatable aptamer sensor targeting 17ß-estradiol for detection of female-specific blood in mixed samples. With the sensor, we were able to detect blood originating from females using a variable light source (495 nm). The sensor was especially sensitive to blood from young females (10-40 years) but not to blood from older females (≥ 50 years). Genomic DNA was extracted from the female blood specimens identified by this method and used for quantification and short tandem repeat genotyping. We confirmed that there was no fluorescence interference from the aptamer sensor. These results indicate that this novel aptamer sensor can be used to analyze evidentiary blood samples and thereby facilitate subsequent genetic identification.

Effect of Benzidine Test on DNA Analysis of Bloodstain / 法医学杂志

Objective To explore the effect of benzidine test and related reagents on DNA analysis of bloodstain. Methods A total of 970 bloodstain filter paper samples with 1μL venous blood were collected, and 10 of them acted as control samples. After benzidine test and related reagent processing, DNA of 960 samples was extracted by Chelex-100 and silica bead methods and then multiplex amplified by AmpF(e)STRTM IdentifilerTM Plus PCR kits. The results of STR typing were compared between different groups. Results DNA were extracted immediately after benzidine test. Totally STR loci (3.80±1.34) were detected by silica bead method, while no STR loci were obtained by Chelex-100 method. Thirteen sam-ples (21.7％) with whole STR typing results were obtained by drying after benzidine test, and the STR locus number (12.90±1.49) which obtained by silica bead method was much higher than by Chelex-100 method (4.70±1.96) (P<0.05). When DNA was extracted immediately after the addition of glacial acetic acid, the STR locus number was (9.40±2.09) by silica bead method, but no STR typing result was obtained by Chelex-100 method. All 15 STR loci could be obtained by only adding glacial acetic acid after drying and only adding tetramethylbenzidine alcoholization liquid or 3％ hydrogen peroxide liquid. Conclusion Benzidine test has significant influence on DNA analysis of bloodstain. The Chelex-100 method is not suitable for the DNA extraction of bloodstain after benzidine test. Drying after benzidine test and silica bead methods can effectively enhance the STR locus number of bloodstain.

[The specific features of the blood stains on the water-lubricated surfaces]. / Osobennosti sledov kapel' krovi na poverkhnosti, smochennoi vodoi.

The objective of the present study was the experimental investigation of the morphological characteristics of the 20 mcl blood droplets which freely fell down onto the surfaces that were either dry or moderately and profusely lubricated with water. The morphological properties of the resulting blood stains on the glass surfaces, dry or moderately and profusely lubricated with water, are described. The most characteristic features of such stains at the wet surface were the uneven serrated and stripe-like prominences, radial 'lucent stripes' passing into the splashing elements. The size of the blood stains at the water-lubricated surface increased by 8-44%.

Effect of anticoagulation therapy on drying times in bloodstain pattern analysis.

In forensic case work, blood stain pattern analysis frequently aids in deducing the chain of actions or parts thereof taking place during an event leading to blood loss. Wiped single blood stains and/or groups of blood stains are seen at a majority of complex crime scenes. The appearance of wiped blood stains depends on droplet volume and stain age (as a function of blood viscosity and the degree of stain skeletonization) and characteristics of the stained surface (i.e., texture, temperature). Furthermore, based on the biochemical and biophysical properties of blood, not only the drying processes, but also complex coagulation cascades are relevant to the assessment of wiped blood stains. This study was designed to determine if anticoagulation therapies markedly affect the wipeability of blood stains over times elapsed since deposition and the overall drying process. A total of 813 blood stains, originating from donors being treated with acetylsalicylic acid (ASA), clopidogrel + ASA, low-molecular-weight heparin, or rivaroxaban, were dropped on common household tiles. Wipeability at an ambient temperature of 20 °C was tested for 22 time periods (1, 2, 3, 5, 10, 15…105 min since deposition). Whereas stains consisting of untreated blood were dried within 55 min, wipeability of all droplets originating from donors with prior anticoagulation treatment showed pronounced delays compared with the control, ranging from 20 min (ASA and clopidogrel + ASA) to 45 min (rivaroxaban). This pronounced effect was not seen in earlier studies, which might be explained by the higher volume of droplets used in this study, which resulted in a shift in relevance from drying to clotting processes. Significant differences between the drying times of the various anticoagulation regimes might be attributed to anticoagulant activity against different targets in the coagulation cascades. In conclusion, anticoagulation treatment prior to blood loss significantly affected the wipeability of blood stains. Anticoagulation therapy should therefore be taken into account in the analysis of blood stain patterns.

Optimizing direct amplification of forensic commercial kits for STR determination.

Direct DNA amplification in forensic genotyping reduces analytical time when large sample sets are being analyzed. The amplification success depends mainly upon two factors: on one hand, the PCR chemistry and, on the other, the type of solid substrate where the samples are deposited. We developed a workflow strategy aiming to optimize times and cost when starting from blood samples spotted onto diverse absorbent substrates. A set of 770 blood samples spotted onto Blood cards, Whatman® 3 MM paper, FTA™ Classic cards, and Whatman® Grade 1 was analyzed by a unified working strategy including a low-cost pre-treatment, a PCR amplification volume scale-down, and the use of the 3500 Genetic Analyzer as the analytical platform. Samples were analyzed using three different commercial multiplex STR direct amplification kits. The efficiency of the strategy was evidenced by a higher percentage of high-quality profiles obtained (over 94%), a reduced number of re-injections (average 3.2%), and a reduced amplification failure rate (lower than 5%). Average peak height ratio among different commercial kits was 0.91, and the intra-locus balance showed values ranging from 0.92 to 0.94. A comparison with previously reported results was performed demonstrating the efficiency of the proposed modifications. The protocol described herein showed high performance, producing optimal quality profiles, and being both time and cost effective.