Validation of a Confirmatory, High Throughput Forensic Body Fluid Assay Using High Performance Liquid Chromatography and Proteomic Mass Spectrometry

Description:
The ever-increasing sensitivity of forensic DNA short tandem repeat (STR) testing, where even a small number of cells can produce to a positive DNA profile, increases the need for a confirmatory body fluid assay in order to determine the source of that DNA. Was the DNA from shed epithelial cells, or from one of the commonly tested forensic body fluids important in criminal investigations – blood, saliva or semen? Such knowledge can add context to a criminal investigation, or to evidence presented in court. Yet currently, no confirmatory assays for these body fluids are routinely employed in public forensic laboratories. The criteria for an ideal confirmatory assay would include, accuracy, sensitivity, scientific soundness, and statistical rigor. The New York City Office of Chief Medical Examiner has developed and validated a confirmatory proteomic mass spectrometry assay for blood, saliva and semen. The assay is based on detection of proteins enriched in these body fluids and whose biological functions are critical to the purpose of these body fluids. To ensure accurate body fluid detection, multiple marker proteins are selected from each body fluid and multiple peptides from each protein. Additionally, by using a multiple reaction monitoring (MRM) mass spectrometry assay, four transition ions from marker peptide confirm each peptide’s identification. Statistical analyses of more than two thousand validation samples were used to determine accuracy, sensitivity, limits of detection, and false positive and negative rates of neat and mock samples of individual and mixed body fluids. Non-targeted human body fluids (vaginal secretions and menstrual blood) were also assayed, as were non-human samples of blood, saliva and semen. This 20-minute HPLC/MRM mass spectrometry assay was designed for high throughput. Reported here are validation results demonstrating the accuracy and sensitivity of this confirmatory forensic body fluid test.

Speaker: Donald Siegel - NYC Office of Chief Medical Examiner
Dr. Siegel received his Ph.D. from the Department of Neuroscience (biochemistry) at the Albert Einstein College of Medicine, and completed his postdoctoral work (molecular biology) at the Rockefeller University. He currently leads the Emerging Technologies Group in the Department of Forensic Biology at the New York City Office of Chief Medical Examiner, evaluating and developing new forensic methods in proteomic mass spectrometry and massive parallel DNA sequencing.