Validation of a Confirmatory Proteomic Mass Spectrometry Body Fluid Assay for Use in Publicly Funded Forensic Laboratories

• NCJ Number: 310765
• Author(s): Donald Siegel
• Date Published: September 2025
• Length: 34 pages
• Grant Number(s): 15PNIJ-21-GG-02712-SLFO
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: Validation of a Confirmatory Proteomic Mass Spectrometry Body Fluid Assay for Use in Publicly Funded Forensic Laboratories

Abstract

DNA short tandem repeat identity testing has become foundational to forensic investigations.  It is scientifically sound, statistically rigorous and above all confirmatory.  Yet as this technology becomes increasingly more sensitive - able to identify an individual from only a few cells - the need to know the source of an individual’s DNA profile is becoming more important.  Currently, however, there are no routinely used confirmatory tests able to identify the source of a person’s DNA from commonly tested forensic sample – e.g. blood, saliva and semen.  Consequently, a suspect may reasonably claim that their DNA profile at a crime scene could be from shed epithelial cells or DNA transfer.  With grant support from the National Institute of Justice, the New York City Office of Chief Medical Examiner has developed, validated and brought into routine casework a confirmatory body fluid assay.  Using liquid chromatography and mass spectrometry, protein markers from each of the above body fluids are used to confirm the presence of a single body fluid or a mixture of body fluids in a forensic sample. For each body fluid, multiple maker proteins were selected based on their essential functions in their respective body fluids. The purpose of this application was to 1) evaluate assay performance metrics of marker detection - e.g. sensitivity, accuracy, precision, etc., using neat, mock, and aged samples, as well as determine how the assay performs with non-targeted body fluids (e.g. menstrual blood and vaginal secretion), 2) determine the likelihood that a body fluid marker peptide may be present in another organism, 3) determine the potential false negative rate due to protein polymorphisms, and 4) establish quality metrics to routinely monitor assay performance.