Improving Seminal Fluid Detection Sensitivity in Extended Post-Coital Intervals by QQQ Mass Spectrometry

As submitted by the proposer: An estimated 84,041 rapes were reported to US law enforcement agencies in 2014, with an increase of 9.6% in 2015. Identification of semen is critical to the analysis of sexual assault samples in forensic laboratories. In cases involving extended post coital reporting, where only trace amounts of male genetic material may remain, the ability to confirm the presence of semen provides critical investigative context. Current methodologies used in forensic laboratories for semen identification suffer from several limitations. Colorimetric assays (e.g., acid phosphatase) require catalytically active enzymes and are not highly specific. Immunochromatographic assays (e.g., RSID Semen and ABAcard p30) are subject to cross-reactivity, non-specific binding and suffer from sensitivity limitations, often failing to detect previously deposited semen just 24 to 33 hours into the post-coital interval. Microscopic identification of spermatozoa is costly and time consuming; does not work for azospermic or vasectomized males, and fails to detect spermatozoa 5+ days post coitus. Alternatively, an assay which combines high-specificity protein biomarkers for seminal fluid with high-sensitivity targeted-ion mass spectrometry has been shown to detect trace levels of seminal fluid results 8+ days into the post coital window. With a level of sensitivity surpassing that possible through Y-STR testing, it becomes important to understand how quantitative levels of semen peptides might correlate with recoverable male DNA. This necessitates converting an already validated qualitative QQQ-MRM assay for seminal fluid into a quantitative one. A quantitative seminal fluid assay could also be used to estimate authentic false positive rates for commercially available immunochromatographic assays by assessing whether the putatively detected target proteins are actually present above the antibody-based detection thresholds. These goals will be met through three core research objectives: (1) Develop and optimize a quantitative QQQ-MRM mass-spectrometry assay using synthetic PSA and Sg I/II proteins to establish a standard curve which can be used to quantitate these proteins in forensic-type samples. (2) Assess the correlation between peptide quantitative values for target seminal fluid peptides and the ability to generate Y-STR profiles from vaginal swabs collected at various post coital intervals. (3) Determine false positive rates associated with immunochromatographic tests of semen-fee vaginal swabs to assess whether target proteins in the sample are actually present above the assay’s sensitivity threshold. The proposed work will improve the quality of sexual assault evidence testing; provide information on the rates of false positive with currently employed seminal fluid assays; allow for the development of more informed interpretation guidelines for seminal fluid identification; enable practitioners to better guide forensic investigations and; facilitate the efficient allocation of resources by allowing analysts to focus downstream genetic analyses on those samples where successful male DNA typing is more likely. Overall, the successful completion and implementation of this research will provide the forensic and criminal justice communities throughout the United States with a powerful tool to aid in the criminal investigation of sexual assault cases. The results of this work will be made publically available in reports to the NIJ, through presentations at professional conferences and in peer reviewed journals. This project contains a research and/or development component, as defined in applicable law. nca/ncf.

An estimated 84,041 rapes were reported to US law enforcement agencies in 2014, with an increase of 9.6% in 2015. Identification of semen is critical to the analysis of sexual assault samples in forensic laboratories.

In cases involving extended post coital reporting, where only trace amounts of male genetic material may remain, the ability to confirm the presence of semen provides critical investigative context.

Current methodologies used in forensic laboratories for semen identification suffer from several limitations. Colorimetric assays (e.g., acid phosphatase) require catalytically active enzymes and are not highly specific. Immunochromatographic assays (e.g., RSID Semen and ABAcard p30) are subject to cross-reactivity, non-specific binding and suffer from sensitivity limitations, often failing to detect previously deposited semen just 24 to 33 hours into the post-coital interval. Microscopic identification of spermatozoa is costly and time consuming; does not work for azospermic or vasectomized males, and fails to detect spermatozoa 5+ days post coitus.

Alternatively, an assay which combines high-specificity protein biomarkers for seminal fluid with high-sensitivity targeted-ion mass spectrometry has been shown to detect trace levels of seminal fluid results 8+ days into the post coital window. With a level of sensitivity surpassing that possible through Y-STR testing, it becomes important to understand how quantitative levels of semen peptides might correlate with recoverable male DNA. This necessitates converting an already validated qualitative QQQ-MRM assay for seminal fluid into a quantitative one. A quantitative seminal fluid assay could also be used to estimate authentic false positive rates for commercially available immunochromatographic assays by assessing whether the putatively detected target proteins are actually present above the antibody-based detection thresholds. These goals will be met through three core research objectives: (1) Develop and optimize a quantitative QQQ-MRM mass-spectrometry assay using synthetic PSA and Sg I/II proteins to establish a standard curve which can be used to quantitate these proteins in forensic-type samples. (2) Assess the correlation between peptide quantitative values for target seminal fluid peptides and the ability to generate Y-STR profiles from vaginal swabs collected at various post coital intervals. (3) Determine false positive rates associated with immunochromatographic tests of semen-fee vaginal swabs to assess whether target proteins in the sample are actually present above the assay’s sensitivity threshold. The proposed work will improve the quality of sexual assault evidence testing; provide information on the rates of false positive with currently employed seminal fluid assays; allow for the development of more informed interpretation guidelines for seminal fluid identification; enable practitioners to better guide forensic investigations and; facilitate the efficient allocation of resources by allowing analysts to focus downstream genetic analyses on those samples where successful male DNA typing is more likely. Overall, the successful completion and implementation of this research will provide the forensic and criminal justice communities throughout the United States with a powerful tool to aid in the criminal investigation of sexual assault cases.

The results of this work will be made publically available in reports to the NIJ, through presentations at professional conferences and in peer reviewed journals.

This project contains a research and/or development component, as defined in the applicable law. nca/ncf