The Food Preferences of the Blow Fly Lucilia cuprina Offered Human Blood,Semen and Saliva,and Various Nonhuman Foods Sources

As human DNA profiles can be obtained from blow fly artifacts, this study aimed to establish the feeding preferences of Lucilia cuprina (Wiedemann) blow flies when offered human biological fluids and nonhuman food sources. One‐day‐old and 3‐day‐old blow flies of both sexes were simultaneously offered human blood, semen and saliva, pet food, canned tuna and honey, and the number and length of visits documented over 6 h. One‐day‐old flies visited pet food and honey most often, but stayed longest on honey and semen. Three‐day‐old flies visited semen and pet food most often, and stayed longest on these food sources. Blood and saliva were the least preferred options for all flies. Overall, flies preferred dry blood and semen to the wet forms. These findings demonstrate that even when other food sources are available, flies at a crime scene may feed on human biological fluids if present, potentially transferring human DNA.     

microRNA Detection in Blood,Urine, Semen,and Saliva Stains After Compromising Treatments

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.     

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection – one per each body fluid type – demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.     

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.     

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.     

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.     

Analysis of the fluorescence of body fluids on different surfaces and times

The use of screening techniques, such as an alternative light source (ALS), is important for finding biological evidence at a crime scene. The objective of this study was to evaluate whether biological fluid (blood, semen, saliva, and urine) deposited on different surfaces changes as a function of the age of the sample. Stains were illuminated with a Megamaxx™ ALS System and photographed with a Canon EOS Utility™ camera. Adobe Photoshop™ was utilized to prepare photographs for analysis, and then ImageJ™ was used to record the brightness values of pixels in the images. Data were submitted to analysis of variance using a generalized linear mixed model with two fixed effects (surface and fluid). Time was treated as a random effect (through repeated measures) with a first-order autoregressive covariance structure. Means of significant effects were compared by the Tukey test. The fluorescence of the analyzed biological material varied depending on the age of the sample. Fluorescence was lower when the samples were moist. Fluorescence remained constant when the sample was dry, up to the maximum period analyzed (60 days), independent of the substrate on which the fluid was deposited, showing the novelty of this study. Therefore, the forensic expert can detect biological fluids at the crime scene using an ALS even several days after a crime has occurred.     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.     

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.     

Advanced statistical analysis of Raman spectroscopic data for the identification of body fluid traces: Semen and blood mixtures

Conventional confirmatory biochemical tests used in the forensic analysis of body fluid traces found at a crime scene are destructive and not universal. Recently, we reported on the application of near-infrared (NIR) Raman microspectroscopy for non-destructive confirmatory identification of pure blood, saliva, semen, vaginal fluid and sweat. Here we expand the method to include dry mixtures of semen and blood. A classification algorithm was developed for differentiating pure body fluids and their mixtures. The classification methodology is based on an effective combination of Support Vector Machine (SVM) regression (data selection) and SVM Discriminant Analysis of preprocessed experimental Raman spectra collected using an automatic mapping of the sample. This extensive cross-validation of the obtained results demonstrated that the detection limit of the minor contributor is as low as a few percent. The developed methodology can be further expanded to any binary mixture of complex solutions, including but not limited to mixtures of other body fluids.     

DNA investigative lead development from blood and saliva samples in less than two hours using the RapidHIT™ Human DNA Identification System

The RapidHIT™ Human DNA Identification System produces full STR DNA profiles (sample in, results out) in 90 min. Here we present results from a new protocol on the RapidHIT System, designed for crime scene samples. Data are presented for blood and saliva samples, with results for a range of samples and substrates. Success rates and sensitivity display a high level of performance for the sample types evaluated in the study and demonstrate the efficacy of the new protocol for crime scene samples.     

Forensic Identification of Blood in the Presence of Contaminations Using Raman Microspectroscopy Coupled with Advanced Statistics: Effect of Sand,Dust, and Soil

Body fluid traces recovered at crime scenes are among the most common and important types of forensic evidence. However, the ability to characterize a biological stain at a crime scene nondestructively has not yet been demonstrated. Here, we expand the Raman spectroscopic approach for the identification of dry traces of pure body fluids to address the problem of heterogeneous contamination, which can impair the performance of conventional methods. The concept of multidimensional Raman signatures was utilized for the identification of blood in dry traces contaminated with sand, dust, and soil. Multiple Raman spectra were acquired from the samples via automatic scanning, and the contribution of blood was evaluated through the fitting quality using spectroscopic signature components. The spatial mapping technique allowed for detection of “hot spots” dominated by blood contribution. The proposed method has great potential for blood identification in highly contaminated samples.     

The detection of gamma globulin and Inv-factors in semen and saliva and of haptoglobins in semen (author's transl)]

Gm- 1-, 2-, and Inv 1-factors can be demonstrated in semen and saliva. For the examination of traces it is necessary to verify the suitable dilutions for the different charges of antisera and to test the eluates of the samples if they have a sufficient concentration. We observed some incorrect negative results in seminal and saliva stains which were apparently caused by insufficient material. The demonstration was independent of the secretor type. Haptoglobin could not be determined in semen and saliva.     

Human semen stain analysis in casework sample by HRM-qPCR

Determining the biological origin of body fluid evidence from crime scenes is extremely important for criminal investigation, especially in sexual assault cases. In Brazil, sexual crimes still present low-resolution rates, where approximately 8% of cases are judged. The determination of the presence of semen in samples from crime scenes as a test prior to DNA analysis is a mandatory requirement in forensic analysis and can help to better understand the dynamics of the event. This report aims to present the methodological strategy used in a criminal case of a home invasion where a t-shirt containing visible stains similar to human semen was found at the site. Convencional tests to detect the presence of PSA and sperm cells were performed on the fabric cutouts which showed negative results. We then processed the fabric samples for genetic analysis after two-years-storage, where were performed automated method for the genetic material isolation (QIAcube, Qiagen). The Real-time PCR analysis were carried out using the SOLIScript 1-step SolisGreen (SolisBiodyne) kit, with specific primers to the TGM4 (Transglutaminase 4) gene, in the Rotor Gene Q-5Plex HRM equipment (Qiagen). The results obtained for the melt curve indicated the presence of human semen in the analyzed samples. After the HRM-qPCR assay we also analyzed the a-STR and Y-STR markers. All the results were useful for the criminal process, which led to the identification of the author of the crime.     

Direct blood group typing of forensic samples using a simple monoclonal antibody assay

A simple direct test for blood group antigens in samples of blood, dried blood, dried blood associated with fabric, semen, vaginal secretions, saliva and fingerprints is described. This test takes advantage of monoclonal antibodies which have been produced in this laboratory, but which are also becoming available commercially in ever increasing numbers. The test is sensitive and reliable as evidenced by its performance in blind studies of more than 700 blood samples. The test requires no special equipment and can be completed in 4 h. The test is sufficiently versatile that new antibodies can be added to the same test format as they become available.     

Applicability of the ParaDNA® Screening System to Seminal Samples

Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA^® Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework.     

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.     

Detection of semen and blood stains using polilight as a light source.

Photoluminescence spectra of dry untreated semen have been measured and a suggested method for rapid detection of untreated semen stains is derived from these measurements. The method is presented in the form of a flow chart to cover most crime scene situations. The absorption spectrum of dry untreated blood has also been measured and a suggested method for enhancement and photography of blood stains is derived from this measurement. The method is presented in the form of a flow chart. Both methods are based on the use of a high intensity light source such as the Polilight.