American forensic DNA practitioners' opinion on activity level evaluative reporting.

The technical advancements made in DNA profiling now allow for very low DNA amounts to be analyzed. Accordingly, the argument often made in criminal courts is not who the DNA belongs to but rather how it was deposited. Despite the complexity of the relevant DNA transfer, persistence, prevalence, and recovery issues, forensic laboratories in some European countries have used evaluative reports with activity level propositions, while this is not current practice in the United States. The purpose of this study was to gain an overview of the opinions about activity level reporting (ALR) held by forensic biologists in the United States. A seventeen-question survey was distributed to members of the American Society of Crime Laboratory Directors and U.S. members of the International Society for Forensic Genetics. The survey included multiple-choice and open-response questions and received 54 responses. The majority of responses expressed moderate support of ALR. Participants mentioned six major concerns to be addressed prior to implementing ALR in the United States: (1) effect of number of variables involved; (2) need of education for practitioners/legal system; (3) inadequate number of activity studies with realistic scenarios; (4) difficulty of achieving admissibility in court; (5) need for standardized approaches/guidelines; and (6) requisite shift in perspective as to the validity of ALR. Overall, this small segment of U.S. forensic DNA practitioners appear to be willing to implement ALR once these concerns are fully addressed and resolved. As a follow-up, it would be worthwhile exploring these and other questions with a larger group and also other disciplines.

High-throughput seminal fluid identification by automated immunoaffinity mass spectrometry.

The identification of semen during a criminal investigation may be a critical component in the prosecution of a sexual assault. Commonly employed enzymatic and affinity-based methods for detection lack specificity, are time-consuming, and only provide a presumptive indication that semen is present where microscopic visualization is unable to meet the throughput demands. Contrary to traditional approaches, protein mass spectrometry provides true confirmatory results, but multiday sample preparation and nanoflow sample separation requirements have limited the practical applicability of these approaches. Aiming at streamlining sexual assault screening by mass spectrometry, the work here coupled a 60-minute rapid tryptic digestion, semenogelin-II peptide affinity purification on an Agilent AssayMap Bravo automation platform, and a 3-minute targeted LC-MS/MS method on an Agilent 6495 triple quadrupole mass spectrometer operating in multiple reaction monitoring mode for detecting semenogelin-II peptides in sexual assault samples. The developed assay was assessed using casework-type samples and was successful in detecting trace levels (0.0001 µl) of semen recovered from both cotton and vaginal swabs, as well as semen recovered from vaginal swabs during menses or adulterated with personal lubricants. This work represents a promising technique for high-throughput seminal fluid identification in sexual assault-type samples by mass spectrometry.

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts.

Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides (GVPs) that contain single-amino-acid polymorphisms to infer the genotype of matching nonsynonymous single-nucleotide polymorphisms for the individual from whom the protein sample originated. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of GVPs increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data-dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data-independent acquisition and a variant of parallel reaction monitoring (PRM) on an Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring (MRM) on an Agilent 6495 triple quadrupole system. In our hands, average GVP detection from a selected panel of 24 GVPs increased from 6.5 ± 1.1 and 3.1 ± 0.8 using data-dependent and -independent acquisition to 9.5 ± 0.7 and 11.7 ± 1.7 using PRM and MRM (p < 0.05), respectively. PRM resulted in a 1.3-fold increase in detection sensitivity, and MRM resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median-estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For PRM and MRM, detected GVPs were validated by the inclusion of stable isotope-labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping and the establishment of validation methods for the evaluation of inferred genotypes.

Developmental validation of a multiplex proteomic assay for the identification of forensically relevant biological fluids.

The aim of this study was to validate a multiplex proteomic assay for the identification of high-specificity protein biomarkers by multiple reaction monitoring mass spectrometry on a triple quadrupole mass spectrometer for the accurate, reliable, and confirmatory identification of bodily fluids commonly encountered in a forensic context. This includes the identification of peripheral blood, semen, saliva, urine, and vaginal/menstrual fluid. The assay is able to efficiently identify pure or mixed stains through the identification of target peptide fragments originating from tissue-specific proteins including: uromodulin from urine; prostatic acid phosphatase, prostate specific antigen and semenogelin-II for semen; statherin, submaxillary gland androgen-regulated protein 3B and amylase for saliva; cornulin, martrigel-induced gene C4 protein, suprabasin and neutrophil gelatinase-associated lipocalin for vaginal/menstrual fluid; and alpha-1 antitrypsin, hemopexin, and hemoglobin subunit beta for peripheral blood. Based on the results of the developmental validation studies which included an assessment of reproducibility and repeatability, sensitivity, species specificity, carryover, mixtures, as well as a series of casework type samples. Only a small selection of case samples was unable to unambiguously identify the target fluid including urine recovered from substrates as well as semen when mixed with personal lubricants. Overall, the mass spectrometry-based workflow offers significant advantages compared to existing serological methods.

Forensic proteomics.

Protein is a major component of all biological evidence, often the matrix that embeds other biomolecules such as polynucleotides, lipids, carbohydrates, and small molecules. The proteins in a sample reflect the transcriptional and translational program of the originating cell types. Because of this, proteins can be used to identify body fluids and tissues, as well as convey genetic information in the form of single amino acid polymorphisms, the result of non-synonymous SNPs. This review explores the application and potential of forensic proteomics. The historical role that protein analysis played in the development of forensic science is examined. This review details how innovations in proteomic mass spectrometry have addressed many of the historical limitations of forensic protein science, and how the application of forensic proteomics differs from proteomics in the life sciences. Two more developed applications of forensic proteomics are examined in detail: body fluid and tissue identification, and proteomic genotyping. The review then highlights developing areas of proteomics that have the potential to impact forensic science in the near future: fingermark analysis, species identification, peptide toxicology, proteomic sex estimation, and estimation of post-mortem intervals. Finally, the review highlights some of the newer innovations in proteomics that may drive further development of the field. In addition to potential impact, this review also attempts to evaluate the stage of each application in the development, validation and implementation process. This review is targeted at investigators who are interested in learning about proteomics in a forensic context and expanding the amount of information they can extract from biological evidence.

Direct seminal fluid identification by protease-free high-resolution mass spectrometry.

Serological screening of sexual assault evidence has traditionally focused on enzyme activity and immunochromatographic assays that provide only a presumptive indication of seminal fluid and have limited sensitivity relative to DNA testing. Seminal fluid detection based on protein mass spectrometry represents a "Next Gen" serological technology that overcomes the specificity and sensitivity limitations of traditional serological screening but requires time-consuming sample preparation protocols. This paper describes a novel "peptidomics" approach to seminal fluid detection that eliminates the need for lengthy trypsin digestion. This streamlines sample preparation to a one-step process followed by high-resolution mass spectrometry to identify naturally occurring seminal fluid peptides and low-molecular weight proteins. Multiple protein biomarkers of seminal fluid were consistently and confidently identified based on the multiplexed detection of numerous endogenous peptides. These included Semenogelin I and II (90% and 86% sequence coverage, respectively); Prostate Specific Antigen/p30 (29% sequence coverage); and Prostatic Acid Phosphatase (24% sequence coverage). The performance of this streamlined peptidomics approach to seminal fluid identification in a forensic context was also assessed using simulated casework samples of the type typically collected as part of a sexual assault examination (e.g., oral and vaginal swabs stained with semen). The resulting data demonstrate that sub-microliter quantities of seminal fluid on cotton swabs can be recovered and reliably detected. This supports the forensic applicability of a peptidomic assay for seminal fluid identification with same-day sample preparation and analysis. Future development and streamlined multiplex peptidomic assays for additional biological stains can easily be envisaged.

Effects of organic acids and common household products on the occurrence of false positive test results using immunochromatographic assays.

Forensic serological analyses often rely on lateral flow immunochromatographic assays to detect proteins that are characteristic of forensically relevant body fluids. In this study, we demonstrate that a positive result, however, is not limited to target protein binding. Citric and lactic acids at various pH levels were tested using 9 different commercial immunochromatographic assays. Varying rates of false positive results were observed with commercial serological assays irrespective of brand or target biological fluid over a wide pH range. The use of kit specific buffers were only partially effective in mitigating the occurrence of organic acid-associated false positive results. Common household products containing organic acids were also tested and found to produce non-specific binding events. This is not to suggest that immunochromatographic assays are not useful as presumptive indicators of bodily fluids. Rather, this study provides a cautionary demonstration of the ease with which organic acids in common household products can generate false positives results. This finding underscores the presumptive nature of these antibody-based lateral flow assay systems.

A survey of senior practitioners regarding most desirable qualifications for hiring and advancement within forensic science.

In today's environment in the field of forensic science where continual advancements in technology and analytical approaches are the norm, the need for forensic practitioners with more specialized and subject-specific knowledge is critical. An updated survey targeting the preferred educational requirements by senior practitioners, crime laboratory directors and managers for entry level applicants was conducted. Results underscored a preference for specialized coursework within specific disciplines in preparing the next generation of forensic scientists while maintaining a strong foundation in the natural sciences at the undergraduate level. Practitioners, regardless of discipline, are seeking applicants with exposure to advanced curriculum content in addition to refined professional skills and critical thinking capabilities. The results of this survey reflect a transition in the needs of crime laboratory employers from a general, broad based criminalistics curriculum as described under current accreditation guidelines, to a focused subject matter rich curriculum with additional management and professional content.