Developmental Validation of RSID™-Saliva: A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Saliva

Abstract:  Current methods for forensic identification of saliva generally assay for the enzymatic activity of α-amylase, an enzyme long associated with human saliva. Here, we describe the R apid S tain ID entification (RSID™-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID™-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID™-Saliva detects less than 1 μL of saliva. The sensitivity of RSID^TM-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID™-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID™-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.     

Messenger RNA Profiling for Forensic Body Fluid Identification： Research and Applications

Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link betw een sample donors and actual criminal acts. How ev-er, the conventional body fluid identification methods are prone to various limitations, such as time con-sumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Re-cently, the analysis of cell-specific messenger RNA expression （mRNA profiling） has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group （EDNAP ） in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profil-ing, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibilitywith current DNA extraction and analysis procedure. In the current review ,we provided an overview of the present know ledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possi-ble practical application to forensic casew ork.     

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.     

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.     

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.     

Optimisation of choline testing using Florence Iodine reagent,including comparative sensitivity and specificity with PSA and AP tests

The detection of semen in forensic science is essential in cases of sexual assault but can be problematic in the absence of spermatozoa. Choline is known to occur in high concentrations in seminal fluid and the Florence Iodine test for its detection has been used in forensic science for many years, however very little is documented regarding its sensitivity and specificity in forensic casework. This paper describes the optimisation of the choline Florence Iodine test (FI) and investigates the sensitivity and specificity of the test against different body fluids, food and drink substances, cleaning products and laboratory chemicals. Comparative testing against Acid Phosphatase (AP) and Prostate Specific Antigen (PSA Seratec®) tests is described and shows that the FI test has greater specificity than the PSA test which cross reacts with a number of body fluids.     

MMP-11多克隆抗体的制备及其法医学意义

目的制备兔抗人基质金属蛋白酶-11(MMP-11)多克隆抗体,建立用MMP-11抗体检测月经血的可行方法,探讨其法医学意义。方法将用基因工程制备的人MMP-11融合蛋白免疫新西兰白兔,饱和硫酸铵法进行抗体纯化。运用蛋白印迹法检测月经血痕、外周血痕、阴道液斑、精液斑、唾液斑和尿液斑,盲测验证该方法的可靠性。结果高效价的兔抗人MMP-11多克隆抗体检测月经血MMP-11蛋白的阳性率为90.48%(93/105),而外周血痕、精液斑、唾液斑、尿液斑和阴道液斑均未检出MMP-11。结论用自制的抗MMP-11多克隆抗体所建立的蛋白印迹法检测月经血中的MMP-11特异性好,灵敏有效,可用于月经血及外周血的鉴别。     

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.     

Highly Sensitive Detection of Blood by Surface Enhanced Raman Scattering,,

Raman spectroscopy for forensic body fluid analysis has received some attention due to the nondestructive nature and potential application for identification at the crime scene; however, its usage has been limited by low detection sensitivity. Surface enhanced Raman scattering (SERS) was evaluated for blood identification for forensic applications. Specifically, a SERS‐active substrate was fabricated, composed of nickel nanotips coated with Ag nanoparticles. Compared with a conventional substrate, the SERS substrate enhanced Raman scattering by more than two orders of magnitude and allowed blood to be identified to a dilution of 1:100,000. Blood was also successfully detected by swabbing the SERS substrate directly on mock evidence. Most importantly, Raman spectra obtained by swabbing the SERS substrate on blood stains were free of luminescence even when blood was deposited on luminescent fabrics. The nondestructive character, simplicity of sample preparation, and high sensitivity make SERS a prime candidate for field and laboratory‐based blood identification.     

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.     

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 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.     

Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids

Raman spectroscopy was used to compare body fluids commonly found at crime scenes in a nondestructive manner. The dry traces of semen, vaginal fluid, sweat, saliva, and blood were analyzed using confocal Raman microscopy with a 785-nm excitation. The results show that the five fluids can be differentiated from one another by visual comparison of their Raman spectra, and that the laser radiation does not damage the sample. The Raman signature of each body fluid is specific and correlates with the known composition of the fluid. Dry traces of human and canine semen exhibited distinctly different Raman signatures. Overall, this preliminary study demonstrates the great potential of Raman spectroscopy for nondestructive, confirmatory identification of body fluids for forensic purposes.     

Screening and identification of tissue-specific methylation for body fluid identification

Identification of body fluid stains can bring important information to crime case. Recent research in epigenome indicates that tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to the type of cell or tissue, which makes it possible to identify body fluid based on analysis of DNA. This study screened and identified tDMRs from genome for forensic purpose. DNA samples from blood, saliva, semen, and vaginal fluid were analyzed by methylation sensitive represent difference analysis and Sequenom Massarray^® quantitative analysis of methylation. Six blood-specific tDMRs were obtained. Two tDMRs display blood-specific hypomethylation, and four tDMRs show blood-specific hypermethylation. These tDMRs may discriminate blood stain from other body fluids. The result indicated that tDMRs could become potential DNA markers for body fluid identification.     

The Use of an Alternate Light Source for Detecting Bones Underwater

When searching underwater crime scenes or disaster scenes for fragmentary human remains, it may be advantageous for forensic divers to be able to detect the presence of bones and teeth among other marine materials (such as shells and rocks). In terrestrial environments, this can typically be accomplished by visual and instrumental methods, but underwater conditions make it difficult to employ detection and sorting techniques in these environments. This study investigates fluorescence of bones and teeth and other marine materials using a submersible alternate light source (ALS) and concludes that an ALS can be a useful tool for detecting bones and teeth in underwater searches as well in terrestrial searches and laboratory environments. The results could impact the methods and equipment used by forensic divers and forensic anthropologists when searching for skeletal remains, potentially increasing the quantity and efficiency of forensic evidence recovered.     

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.     

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.     

In depth investigation of the capabilities and limitations of combined proteomic-MALDI MS based approach for the forensic detection of blood

For the past 7 years, Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) based methods have been developed and published for the forensic detection of blood in stains and fingermarks. However, in the view of adoption in an operational context, further investigation into the capabilities and limitations of this approach must be conducted. The refinement and testing of this approach must also be tailored to the requirements of the end users, enabling them to address the specific circumstances most encountered in a forensic scenario. The present study delves deeper into the assessment of the applicability of MALDI MS based strategy for the reliable and robust detection of human blood through: (i) a semi-qualitative assessment of the sensitivity of the method, (ii) a wider investigation of the compatibility of the method with the prior application of commonly used presumptive tests and (iii) assessment of the specificity of the method (when blood is present in mixture with other biofluids) and of its robustness, by assessing blood detection from a range of porous materials. The findings strengthen the evidence supporting the adoption of MALDI MS based approaches as a confirmatory test for the forensic detection of human blood in an operational context.     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.