Comparative Assessment of Emerging Technologies for Body Fluid Identification

Under previous NIJ awards, targeted Next Generation Serological assays for human body fluids have been developed leveraging emerging technologies such as mRNA sequencing, epigenetics, and proteomics. While an mRNA body fluid assay was the basis for a collaborative exercise between participating EUROFORGEN and EDNAP laboratories, no such study to date has compared these emerging serological assays in order to test their efficiency related to each other as well as against contemporary immunochromatographic tests. This proposed study will leverage collaborations amongst four participating laboratories in order to compare the sensitivity and specificity for each serological workflow. In consultation with the Applied Genetics Group at NIST, homogeneous samples containing body fluid stains of various human origin will be generated, mitigating potential variability from front-end sampling techniques so as to directly compare results among laboratories and methodologies. Samples will be prepared in two phases. In phase 1, semen, venous blood, saliva, and vaginal fluid/menstrual blood will be prepared in triplicate across two concentrations, allowing for system alignments for analytical protocols and internal interpretation guidelines. All samples will be simultaneously analyzed using a traditional capillary electrophoretic workflow for STR analysis, demonstrating compatibility of all body fluid identification workflows with current STR typing technologies. Blind mock casework samples — including degraded biological samples, mixtures commonly encountered in forensic scenarios, samples treated with chemical insult, dilutions of single-source biological samples, and non-target/non-human samples — will be prepared and tested in Phase 2. Instances where samples generate discordant results across methodologies will be identified and evaluated to ascertain why discrepant results were obtained. Samples from both phases will be analyzed by caseworking analysts using RSID™-Blood, RSID™-Semen, and RSID™-Saliva (Independent Forensics) as well as the Menstrual Blood PMB Test (Seratec). Using methodologies developed under previous NIJ funding, researchers will conduct mRNA sequencing using NGS body fluid identification assays developed on both the MiSeq (Illumina) and Ion S5 (ThermoFisher Scientific) platforms. Proteomic analysis will be performed utilizing the 6495 triple quadrupole mass spectrometer (QQQ) coupled to a 1290 ultra-high-performance liquid chromatography system (Agilent Technologies) for mass analysis, while epigenetic analysis will be performed using a Q48 Autoprep Pyrosequencer (QIAGEN). This proposal will help align research surrounding emerging techniques for body fluid identification with operational forensic laboratory needs, while generating valuable information to assist with the technology transfer associated with emerging body fluid identification, helping better inform future research directions. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF