Developmental Validation of a High-Specificity Multiplex Assay for Human Body Fluid Identification

While DNA profiling makes it possible to individualize biological stains, the identification of the stain itself can present forensic serologists with a significant challenge. For example, if a genetic profile generated from a swab of a male suspect's finger is consistent with that of an alleged female victim of sexual assault, more than one interpretation is possible. Did the male sexually assault the victim by digitally penetrating her? Or, did the female lick the suspect's finger at some point - leaving her DNA behind? The ability to reliably determine whether the suspect's finger contains traces of saliva and/or vaginal secretions could enable investigators to better evaluate such alternate interpretations. Current antibody and enzyme activity-based assays used by forensic practitioners for biological stain identification yield only presumptive results. Positive results with non-target body fluids or cross-reactivity with non-human sources have been well documented. Some tests consume unacceptable quantities of precious evidence while for other body fluids there are simply no available tests at all. There is clear value in developing more accurate and standardized approaches for identifying biological stains. The proposed research work is focused on the use of protein biomarkers and mass spectrometry for the confirmatory identification of biological stains. Under awards 2006-DN-BX-K001 and 2009-DN-BX-K165, high-specificity protein biomarker panels were compiled and tested for the accurate, reliable and confirmatory identification of bodily fluids commonly encountered in a forensic context. The research outlined here expands on prior NIJ-supported work by developing and rigorously testing a faster, more accurate, and more sensitive multiplex assay for the simultaneous identification of saliva, semen and vaginal secretions. The assay is designed to efficiently handle either pure or mixed stains. The strategy to achieve this is based on the use of a triple quadrupole mass spectrometer (QQQ) utilizing the multiple reaction monitoring (MRM) technique. This instrument utilizes a two-fold ion filtration method where peptides and their subsequent fragmentation ions are accurately detected even against the complex background of non-target molecules present in any body fluid. This two-fold filtration allows for very high selectivity, specificity, and multiplexing capabilities. The specific goal of the current proposal is to fully develop a QQQ multiplex by incorporating the remaining biomarkers into the assay, thoroughly assess its performance limits and thus its potential applicability to casework. This goal will be achieved through the following four core research objectives: 1) Select diagnostic target ions for the six-stain multiplex QQQ assay. 2) Thoroughly optimize the performance of the six-stain multiplex QQQ assay. 3) Conduct a rigorous developmental validation that meets the FBI's QAS standards. 4) Develop appropriate and reliable SOPs and Interpretation Guidelines. A prototype QQQ-MRM three-stain multiplex assay has already been designed, developed and tested by the Principle Investigators research team. The assay accurately and unambiguously discriminates between seminal fluid, saliva, and vaginal secretions with an instrument run time of only 11 minutes. This multiplex assay yields confirmatory results for the identification of human seminal fluid, saliva, and vaginal secretions. This work has the potential to significantly improve the accuracy and sensitivity of forensic serological testing. It will provide practitioners with greatly improved tests for saliva and seminal fluid while also enabling the identification of vaginal secretions for which there are currently no accurate tests. The multiplex design will eliminate the need to perform separate tests on an unknown stain. In short, the successful completion and implementation of this research will provide the forensic community with a powerful tool. ca/ncf