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.     

Infrared Imaging of the Crime Scene: Possibilities and Pitfalls

All objects radiate infrared energy invisible to the human eye, which can be imaged by infrared cameras, visualizing differences in temperature and/or emissivity of objects. Infrared imaging is an emerging technique for forensic investigators. The rapid, nondestructive, and noncontact features of infrared imaging indicate its suitability for many forensic applications, ranging from the estimation of time of death to the detection of blood stains on dark backgrounds. This paper provides an overview of the principles and instrumentation involved in infrared imaging. Difficulties concerning the image interpretation due to different radiation sources and different emissivity values within a scene are addressed. Finally, reported forensic applications are reviewed and supported by practical illustrations. When introduced in forensic casework, infrared imaging can help investigators to detect, to visualize, and to identify useful evidence nondestructively.     

RNA/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.     

Detection and visualization of human tears using alternate light sources for forensic purposes

The current scientific techniques for locating body fluids focus on quick and effective methodologies for easy and reliable identification. Efficient detection and identification of body fluids play a vital role in establishing the ‘corpus delecti’ of a crime. Non-destructive techniques such as the use of Alternate Light Sources (ALS) have been exploited for crime scene searches over large areas and detection of body fluids such as blood, semen, vaginal secretions, and saliva on a range of substrates. Tears are rarely found but can be considered as potential body fluid evidence due to their unique biochemical and molecular properties. Tears are secreted in response to physical or emotional stimuli. Due to the small volume of secretions, they are often overlooked in the crime scene. Tears may be found on surfaces such as clothing, bedding, tissue, handkerchief, or balaclava. The use of ALS to locate tears on tissue paper and fabric surfaces was tested which were not apparent to the naked eye. Tears stains were successfully detected on surfaces of forensic interest with varying sample ages up to three months with a broad excitation spectrum between 254 nm and 410 nm. Dried stains on tissue paper and fabric substrates were better detected with sharp margins, clear stain pattern visibility, and fluorescence intensity in comparison with moist and fresh stains. Tears stains can hence be detected with the use of ALS and suitable filter combinations under normal conditions and do not require any specific settings to locate them. These findings are suggestive for easy and quick identification of tears on large surfaces and as a presumptive test for forensic casework evidence examination.     

A comparison of the sensitivity of typing group-specific component (Gc) and the phosphoglucomutase (PGM1) locus in control and casework bloodstains by three isoelectric focusing methods

The performance of typing group-specific component (Gc) in bloodstains by two isoelectric focusing methods followed by its detection with silver staining has been compared with an established forensic system of typing phosphoglucomutase (PGM1) locus phenotypes by isoelectric focusing (IEF) in 1 mm gels. For Gc typing ultra-thin isoelectric focusing (UTIEF) gels and immobilized pH gradient (IPG) gels were used. Both laboratory prepared stains and casework stains were examined. The Gc UTIEF method is approximately eight times more sensitive than the existing PGM1 1 mm IEF method for control and casework stains. However, on average, a larger amount of stain was taken from casework stains than control stains for each typing system. A total of 53 casework stains were examined. Comparable success rates of 62% and 64% were obtained for typing Gc on UTIEF gels and PGM1 by 1 mm IEF, respectively. A success rate of 55% was obtained for typing Gc on IPGs. Bloodstains that were over 200 days old were successfully grouped by all three methods.     

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).     

An Analysis of Hounsfield Unit Values and Volumetrics from Computerized Tomography of the Proximal Femur for Sex and Age Estimation

Sex and age are two elements in the establishment of a biological profile for forensic identification. While the pelvic bones are the most ideal structures for sex estimation, the condition of a body is not always ideal due to the nature of death, such as in mass disasters, or postmortem processes. This study utilized CT scans and resultant 3D models of 100 male and 100 female adults of known ages ranging from 18 to 98 years old to collect volumetric and Hounsfield unit measurements of the proximal femur. Equations were created to establish logistic regression models for sex estimation and linear regression models for age estimation. The resultant sex estimation method had an accuracy of 93.5% and utilized the volume of the proximal femur. This study provides three linear regression models for age with an accuracy range of 86%–92% ±12 years. As imaging technologies are increasingly adopted for forensic purposes, the power of 3D data will provide the opportunity for more quantitative and reproducible analyses. The proposed method for sex and age estimation provides a reliable tool that can be utilized in both day-to-day casework and disaster victim identification.     

ABO基因型检验及其法医学应用研究

目的采用ＰＣＲ技术对ＡＢＯ血型系统进行基因型检验。方法选择最佳扩增条件进行四引物复合扩增,用限制性内切酶ＫｐｎＩ和ＡｌｕＩ分别酶解扩增产物,电泳分离、银染显色法检验ＡＢＯ基因型。结果对２７０例血斑、２０例混合斑、２０根毛发（有毛囊）、１２份唾液斑等不同的生物检材进行了分型,与血清学方法检验结果相符。结论该方法能够应用于法医学的检验     

Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.     

Routine phenotyping of native and desialyzed alpha 2HS-glycoprotein from blood stains

The application of a polyacrylamide gel isoelectric focusing (PAGIEF) and immunoblotting procedure for the identification of native alpha 2HS-glycoprotein (AHSG) in routine casework blood stains has produced reportable results on 57.2% of samples. This reporting rate is lower than that for group specific component (GC) (83.8%) and phosphoglucomutase (PGM 1) (72.8%) phenotyping of the same samples. Blood stain samples were desialyzed with 1 U/ml neuraminidase, overnight at room temperature prior to PAGIEF in gels containing pharmalyte pH 5-6 and 2.5 M urea. Simple AHSG patterns were developed by immunoblotting. This procedure was five times as sensitive as the native AHSG method and desialyzation was reproducible over a range of incubation times and neuraminidase concentrations. The application of the desialyzed AHSG analysis to routine casework samples has resulted in a significant increase in the number of reportable results (762 reported out of 1027 samples). This reporting rate (74.2%) compares favourably with that for GC (79.1%) and PGH 1 (69.6%) phenotyping of the same samples. The three AHSG alleles (AHSG*1, 2 and 3) are clearly resolved after sample desialyzation and separation in gels containing pharmalyte pH 5-6 and 2.5 M urea. The sensitivity of desialyzed AHSG phenotyping approaches that of GC and this technique is worthy of inclusion in blood stain screening protocols of forensic laboratories in regions where the population has a limited range of rare AHSG alleles.     

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.     

Interpretation of ante-mortem stature estimates in South Africans

We developed a simple method for animal species identification of humans, dogs and cats, using a multiplex single-base primer extension reaction in the cytochrome b gene. Using this method, three points of a single nucleotide in the cytochrome b gene were examined in these species using primers of different lengths. Our method was found to be able to successfully identify humans (26 samples), dogs (21 samples) and cats (9 samples), and no differences were found among the samples from each animal species in this study. The amount of template DNA required was over 0.01 ng for humans and dogs, and over 0.1 ng for cats. The present method was able to identify animal species from hair shaft (2 cm) and forensic casework samples (blood stains and hair shafts), and is thus a useful tool for animal species (human, dog and cat) identification in forensic science.     

A novel method for the identification of saliva by detecting oral streptococci using PCR

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva. Our data indicated that S. salivarius is more reliable than S. mutans as an indicator of saliva presence, because the detection rates for S. salivarius and S. mutans by this method were 100% and 90%, respectively. Furthermore, S. salivarius was detected in all saliva stain samples, whereas S. mutans was only identified in 60% of the stains. Finally, using this method we were able to successfully detect S. salivarius and S. mutans in mock forensic samples. We therefore suggested that this method is useful for the identification of saliva in forensic science.     

Estimating age of humans based on telomere shortening

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

Remote spectroscopic identification of bloodstains

Blood detection and identification at crime scenes are crucial for harvesting forensic evidence. Unfortunately, most tests for the identification of blood are destructive and time consuming. We present a fast and nondestructive identification test for blood, using noncontact reflectance spectroscopy. We fitted reflectance spectra of 40 bloodstains and 35 nonbloodstains deposited on white cotton with spectroscopic features of the main compounds of blood. Each bloodstain was measured 30 times to account for aging effects. The outcome of the blood measurements was compared with the reflectance of blood-mimicking stains and various body fluids. We found that discrimination between blood and nonblood deposited on white cotton is possible with a specificity of 100% and a sensitivity of 98%. In conclusion, a goodness of fit between the sample's reflectance and the blood component fit may allow identification of blood at crime scenes by remote spectroscopy.     

Use of styryl 11 and STaR 11 for the luminescence enhancement of cyanoacrylate-developed fingermarks in the visible and near-infrared regions

In current casework, most post-cyanoacrylate stains rely on luminescence emission in the visible region (400-700 nm). While traditional stains such as rhodamine 6G work well under most circumstances, some surfaces may generate background luminescence under the same conditions. Detection in the near-infrared region (NIR > 700 nm) has shown to be effective in minimizing the interferences from such surfaces. The laser dye styryl 11 generated strongly luminescent fingermarks when applied after cyanoacrylate fuming on all surfaces tested. When compared to rhodamine 6G, the dye was superior only when viewed in the NIR. Styryl 11 was subsequently combined with rhodamine 6G, and the mixed stain formulation (named StaR 11 by the authors) induced stronger luminescence compared with styryl 11 alone with an ability to visualize in both the visible and NIR regions. Reliable and consistent results were obtained when using either styryl 11 alone or the STaR 11 mixture. The enhancement achieved did not otherwise vary depending on the source of the fingermark secretions. With visualization possible in both the visible and NIR regions, the styryl 11/rhodamine 6G mixture showed significant potential as a post-cyanoacrylate stain.     

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)     

Comparison of methods for visualizing blood on dark surfaces

Difficulties can arise when screening dark casework items for blood, a poor contrast between blood and the background can mean stains are not always evident. Typical indirect searching methods can be time consuming and may result in potentially important bloodstains being missed. Luminol, fluorescein, hydrogen peroxide, ultraviolet light and infrared photography were tested in an effort to find a rapid and efficient blood search tool for direct application to dark surfaces. Methods were compared in their sensitivity, specificity, ability to work on various surface types and their effect on DNA extraction and typing. Along with experimental results, the ease of use, costs and the health and safety considerations were also compared. Hydrogen peroxide was determined to be the most effective method. However, where blood was likely to be dilute, luminol was proposed due its greater sensitivity.     

Specimen preparation for ABO determination of various kinds of blood stains

Trying to optimize the preparation of blood stains, we found methanol fixation not to produce very good results for the determination of ABO blood group antigens. It is advantageous to transfer blood stains before testing to cotton cloth. This transfer is also of practical use if blood stains are to be saved on a smooth surface for lateral determination. We testet on 35 different carrier materials, on which blood stains in casework often were found, whether blood grouping gave better results on either the original material or after transfer. Results are shown on a table. The test revealed, that solubility of the stain in aqua dest is a good sign for a successful transfer. Blood stains on pine-wood soil, soil and loam were not suited for ABO grouping.     

Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the extraction of forensic casework samples

Robotic systems are commonly utilized for the extraction of database samples. However, the application of robotic extraction to forensic casework samples is a more daunting task. Such a system must be versatile enough to accommodate a wide range of samples that may contain greatly varying amounts of DNA, but it must also pose no more risk of contamination than the manual DNA extraction methods. This study demonstrates that the BioMek 2000 Laboratory Automation Workstation, used in combination with the DNA IQ System, is versatile enough to accommodate the wide range of samples typically encountered by a crime laboratory. The use of a silica coated paramagnetic resin, as with the DNA IQ System, facilitates the adaptation of an open well, hands off, robotic system to the extraction of casework samples since no filtration or centrifugation steps are needed. Moreover, the DNA remains tightly coupled to the silica coated paramagnetic resin for the entire process until the elution step. A short pre-extraction incubation step is necessary prior to loading samples onto the robot and it is at this step that most modifications are made to accommodate the different sample types and substrates commonly encountered with forensic evidentiary samples. Sexual assault (mixed stain) samples, cigarette butts, blood stains, buccal swabs, and various tissue samples were successfully extracted with the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System, with no evidence of contamination throughout the extensive validation studies reported here.