DNA testing can link an item of evidence to an individual but it cannot identify the biological fluid from which the genetic material originated. Such basic information, however, is often critical to identifying probative evidence in a criminal investigation. It is not currently possible for forensic scientists to unambiguously identify forensically-relevant body fluids with existing assays which yield only presumptive results and are often not amenable to trace or degraded samples. In fact, for some body fluids (e.g., vaginal fluid and menstrual blood) there are no commercial tests available.
In stark contrast to historical methods, modern protein mass spectrometry-based technologies make it possible to rapidly obtain a confirmatory identification of even trace quantities of human body fluids through the direct detection of fluid-specific proteins. Currently, analysis of whole-proteome maps for six forensically relevant biological fluids (i.e., peripheral and menstrual blood, vaginal fluid, urine, saliva and seminal fluid) have identified biomarkers for each body fluid through prior support for basic research from the National Institute of Justice. The target body fluid specificity of each biomarker and the reliability of its detection have since been confirmed in a larger human population sample by quadrupole time-of-light (QTOF) mass spectrometry. This workflow has been developed and validated using our state of the art instrumentation in partnership with Agilent Technologies®. This prototype assay accurately identifies trace amounts of human body fluids in both single and mixed source samples, however, longer run times and manual sample preparation makes it not ideally suited for criminal casework. The current research focus for this project is to deploy laboratory-ready assays for use in forensic laboratories. This aim in being accomplished through grant funded research as well as collaborations with external forensic laboratories.
Developing High Throughput Capabilities
Though funding awarded by the United States Department of Defense a robotic sample preparation with high throughput triple quadrupole mass spectrometry will be integrated into the workflow in order to develop a simple, reliable, and streamlined assay for deployment in caseworking laboratories. In transferring the prototype assay to a high speed triple quadrupole mass spectrometry system, operational parameters will be reoptimized and undergo rigorous validation studies. The final automated samples processing multiplex assay will be implemented in a forensic laboratory to assess the critical functionalities on the method. In collaboration with Agilent Technologies and the Brasilia Civil police, a mass spectrometry-based based workflow has already been deployed and successfully applied to authentic case samples. This work is helping to provide law enforcement and forensic laboratories with reduced cost screening with accurate and confirmatory front end identification of evidentiary stains of interest.
Application to Sexual Assault Kit Analysis
Approximately 18% of women in the United States have been raped in their lifetime, including 1.8 million adolescents. The timely recovery of physical evidence is critical to investigations of sexual assault. Unfortunately, many sexual assault victims delay reporting an incident several days after an attack, especially in cases involving child victims. As the time between an assault and evidence collection increases, the ability to successfully recover seminal fluid, sperm cells and/or DNA rapidly diminishes. Commercial kits used in forensic laboratories to detect seminal fluid often fail to detect semen just 24 to 33 hours into the post-coital interval, indicating the need for a more sensitive and specific assay for the identification of seminal fluid. Applying protein mass spectrometry-based technologies, a monoplex method developed and optimized for the sole identification of seminal fluid provides the opportunity to further enhance the sensitivity of a seminal fluid assay. This has the potential to extend the post-coital interval for successful detection of trace seminal fluid, providing a powerful tool for the investigation of sexual assault. In partnership with NMS Labs in Willow Grove, PA, this methodology is being incorporated into a novel sexual assault backlog workflow designed to screen and prioritize sexual assault type samples.
Publications and Presentations
Discovery of highly specific protein markers for the identification of biological stains
Kevin Legg, Roger Powell, Nichole Reisdorph, Rick Reisdorph and Phillip Danielson. Electrophoresis. 2014; 35 (21-22): 3069-3078.
Body Fluid Identification by Mass Spectrometry – 2015
Kevin Legg and Phillip Danielson. Green Mountain Meeting, Platform Presentation 2015
Body Fluid Identification from Sexual Assault Evidence
Kevin Legg, Heather McKiernan, Phillip Danielson, Christian Westring. AAFS Poster Presentation 2015
Development and Testing of a Mass Spectrometry Based Assay for the Identification of Biological Stains
Kevin Legg, Heather Mazzanti (McKiernan), Phillip Danielson, Christian Westring. AAFS Platform Presentation 2014