Use of Novel Chemistry & Microwave Instrumentation to ImproveBody Fluid Assay Sensitivity & Speed while Reducing Costs

Body fluid identification plays a vital role in forensic investigations contributing to both case evidence as well as directing the course of further investigations - particularly with respect to sexual assaults. Yet the methods used for body fluid detection have, for the most part, failed to keep pace with scientific and technological advances. As a consequence, current testing is a diverse mixture of techniques that vary dramatically in sensitivity, reproducibility, speed and cost. Most are not confirmatory, and there are many body fluids for which no routine tests are currently available, e.g. menstrual blood and vaginal fluid. With the aid of previous NIJ support, we developed a proteomic mass spectrometry (MS) assay for blood, saliva and semen. This single test is confirmatory and can automatically identify any one of these body fluids in an unknown sample without a priori knowledge of what they might be. Species identification is also automatically determined. Our current system can process ~ 750 samples per week (see 2.3 Preliminary Results). Many more samples could be processed, but trypsin digestion of proteins, a prerequisite for nearly all MS assays, requires overnight incubation. We have also developed confirmatory assays for menstrual blood, vaginal fluid as well as body fluid mixtures. These tests, however, require protein purification prior to MS and consequently require significantly more time. Recently, advances in protein chromatography and microwave technology (see Research Design & Methods) have made significant gains not only in dramatically reducing sample preparation and digestion times (in some cases from days to minutes), but also improving the quality of analytes and consequently, test results. In 2009 the New York City Office of Chief Medical Examiner processed a total of 68,159 samples for body fluid identification (11,756 Kastle-Meyer presumptive blood tests, 6,961 amylase presumptive saliva tests, and 12,035 AP presumptive and 12,719 sperm searches or p30 tests. This type of consecutive testing is time and sample consuming, and expensive. We are beginning validation testing of our MS assay. The overarching goal of this application is to test and evaluate these new technologies and integrate them into our current assays in order to make them faster, more accurate, more sensitive and less expensive. In addition, we plan to evaluate the simultaneous extraction of protein and DNA from samples in order to conserve sample use, as well as incorporate liquid handling robotics into our assay workflow. We believe that the proposed work directly addresses both the applied research and developmental goals outlined in the NIJ solicitation. With regard to applied research, our work will systematically use scientific methods to evaluate the chemical properties of body fluid marker proteins with respect to their ability to bind to and be enriched by a novel protein chromatography matrix, which, if successful, will improve marker detection sensitivity while reducing assay time and costs. With regard to developmental goals, this work aims to integrate new chemistries and instrumentation into a single, semi-automated high-throughput robotic assay system for the detection of both simple (blood, saliva and semen) and complex (menstrual blood & vaginal fluid) body fluids as well as body fluid mixtures. If successful, this assay system will be more sensitive and less expensive than current methods, and we believe become the standard for body fluid testing. ca/ncf