Neurophysiological outcomes that sustained clinically significant improvements over 3 years of physiologic ECAP-controlled closed-loop spinal cord stimulation for the treatment of chronic pain.

INTRODUCTION: A novel, spinal cord stimulation (SCS) system with a physiologic closed-loop (CL) feedback mechanism controlled by evoked compound action potentials (ECAPs) enables the optimization of physiologic neural dose and the accuracy of the stimulation, not possible with any other commercially available SCS systems. The report of objective spinal cord measurements is essential to increase the transparency and reproducibility of SCS therapy. Here, we report a cohort of the EVOKE double-blind randomized controlled trial treated with CL-SCS for 36 months to evaluate the ECAP dose and accuracy that sustained the durability of clinical improvements. METHODS: 41 patients randomized to CL-SCS remained in their treatment allocation and were followed up through 36 months. Objective neurophysiological data, including measures of spinal cord activation, were analyzed. Pain relief was assessed by determining the proportion of patients with ≥50% and ≥80% reduction in overall back and leg pain. RESULTS: The performance of the feedback loop resulted in high-dose accuracy by keeping the elicited ECAP within 4µV of the target ECAP set on the system across all timepoints. Percent time stimulating above the ECAP threshold was >98%, and the ECAP dose was ≥19.3µV. Most patients obtained ≥50% reduction (83%) and ≥80% reduction (59%) in overall back and leg pain with a sustained response observed in the rates between 3-month and 36-month follow-up (p=0.083 and p=0.405, respectively). CONCLUSION: The results suggest that a physiological adherence to supra-ECAP threshold therapy that generates pain inhibition provided by ECAP-controlled CL-SCS leads to durable improvements in pain intensity with no evidence of loss of therapeutic effect through 36-month follow-up.

ECAP-controlled closed-loop versus open-loop SCS for the treatment of chronic pain: 36-month results of the EVOKE blinded randomized clinical trial.

INTRODUCTION: The evidence for spinal cord stimulation (SCS) has been criticized for the absence of blinded, parallel randomized controlled trials (RCTs) and limited evaluations of the long-term effects of SCS in RCTs. The aim of this study was to determine whether evoked compound action potential (ECAP)-controlled, closed-loop SCS (CL-SCS) is associated with better outcomes when compared with fixed-output, open-loop SCS (OL-SCS) 36 months following implant. METHODS: The EVOKE study was a multicenter, participant-blinded, investigator-blinded, and outcome assessor-blinded, randomized, controlled, parallel-arm clinical trial that compared ECAP-controlled CL-SCS with fixed-output OL-SCS. Participants with chronic, intractable back and leg pain refractory to conservative therapy were enrolled between January 2017 and February 2018, with follow-up through 36 months. The primary outcome was a reduction of at least 50% in overall back and leg pain. Holistic treatment response, a composite outcome including pain intensity, physical and emotional functioning, sleep, and health-related quality of life, and objective neural activation was also assessed. RESULTS: At 36 months, more CL-SCS than OL-SCS participants reported ≥50% reduction (CL-SCS=77.6%, OL-SCS=49.3%; difference: 28.4%, 95% CI 12.8% to 43.9%, p<0.001) and ≥80% reduction (CL-SCS=49.3%, OL-SCS=31.3%; difference: 17.9, 95% CI 1.6% to 34.2%, p=0.032) in overall back and leg pain intensity. Clinically meaningful improvements from baseline were observed at 36 months in both CL-SCS and OL-SCS groups in all other patient-reported outcomes with greater levels of improvement with CL-SCS. A greater proportion of patients with CL-SCS were holistic treatment responders at 36-month follow-up (44.8% vs 28.4%), with a greater cumulative responder score for CL-SCS patients. Greater neural activation and accuracy were observed with CL-SCS. There were no differences between CL-SCS and OL-SCS groups in adverse events. No explants due to loss of efficacy were observed in the CL-SCS group. CONCLUSION: This long-term evaluation with objective measurement of SCS therapy demonstrated that ECAP-controlled CL-SCS resulted in sustained, durable pain relief and superior holistic treatment response through 36 months. Greater neural activation and increased accuracy of therapy delivery were observed with ECAP-controlled CL-SCS than OL-SCS. TRIAL REGISTRATION NUMBER: NCT02924129.

Real World Clinical Utility of Neurophysiological Measurement Utilizing Closed-Loop Spinal Cord Stimulation in a Chronic Pain Population: The ECAP Study Protocol.

Background: Spinal cord stimulation (SCS) is an established chronic pain treatment, but the effectiveness of traditional, open-loop paradigms has been plagued by variable sustainability in a real-world setting. A new approach, utilizing evoked compound action potential (ECAP) controlled closed-loop (CL) SCS, continuously monitors spinal cord activation and automatically adjusts the stimulation amplitude of every pulse, maintaining stimulation at the prescribed ECAP level through this continual feedback mechanism. Recent studies demonstrated the long-term safety and efficacy of ECAP-controlled CL-SCS. Here, we report the design of a prospective, multicenter, single-arm feasibility study to characterize clinical outcomes in a real-world chronic pain population utilizing ECAP-controlled CL-SCS. Objective neurophysiological measurements such as device performance and patient therapy compliance, will be analyzed against baseline biopsychosocial assessments, to explore the clinical utility of these objective physiologic biomarkers in patient phenotyping. Methods: This study will enroll up to 300 subjects with chronic, intractable trunk and/or limb pain in up to 25 United States investigation sites. Subjects meeting eligibility criteria will undergo a trial procedure and a permanent implant following a successful trial. Neurophysiological measurements (measured in-clinic and continuously during home use) and clinical outcomes including pain, quality-of-life, psychological, emotional, and functional assessments will be collected at baseline, trial end, and up to 24-months post-implantation. Discussion: Associations between objective neurophysiological data, clinical evaluation and patient-reported outcomes may have important clinical and scientific implications. They may provide novel insights about the chronic pain pathophysiology, its modulation during CL-SCS, and identification of pain phenotypes and/or mechanisms associated with treatment response during SCS trials and long-term therapy. Data from the ECAP study could lead to improvements in diagnosis, assessment, patient identification and management of chronic pain. It could also provide the foundation for development of a new SCS treatment approach customized by the patient's pain phenotype, unique neurophysiology, and disease severity.

Body Fluid Identification in Samples Collected after Intimate and Social Contact: A Comparison of Two mRNA Profiling Methods and the Additional Information Gained by cSNP Genotypes.

The ability to associate a contributor with a specific body fluid in a crime stain can aid casework investigation. The detection of body fluids combined with DNA analyses may supply essential information, but as the two tests are independent, they may not be associated. Recently, the analysis of coding region SNPs (cSNPs) within the RNA transcript has been proven to be a promising method to face this challenge. In this study, we performed targeted RNA sequencing of 158 samples (boxershorts, fingernail swabs and penile swabs) collected from 12 couples at different time points post-intimate contact and after non-intimate contact, using the Ion S5™ System and BFID-cSNP-6F assay. The aim of the study was to compare the performance of the MPS and CE methods in the detection of mRNA markers, and to associate body fluids with contributors by their cSNP genotypes. The results of the study show a lower success rate in the detection of vaginal mucosa by the MPS compared to the CE method. However, the additional information obtained with the cSNP genotypes could successfully associate body fluids with contributors in most cases.

Targeted S5 RNA sequencing assay for the identification and direct association of common body fluids with DNA donors in mixtures.

The evidentiary value of DNA profiles varies depending upon the context in which the DNA was found. Linking a DNA profile to a particular cellular phenotype in mixtures may aid in assessing its evidentiary relevance and value. We report the development of two dual-function high-resolution messenger RNA (mRNA) sequencing assays that can each identify the presence of 6 body fluids/tissues (blood, semen, saliva, vaginal secretions, menstrual blood, skin) and, via coding region SNPs (cSNPs) present in the body fluid-specific mRNA transcripts, directly associate particular body fluids with their specific DNA donors in mixtures. The original blood, semen, and saliva (BSS) assay contains 23 cSNPs for blood, semen, and saliva, while the expanded 6F (all 6 fluids/tissues) assay encompasses the BSS assay and also contains 23 additional cSNPs for vaginal secretions, menstrual blood, and skin. Software tools were developed to infer the identity of the body fluids present as well as providing the corresponding cSNP genotypes. Concomitant genomic DNA assays (BSS-d and 6F-d), required to genotype the same cSNPs from persons of interest/inferred contributors to the body fluid mixture, were also developed. Body fluid specificity was demonstrated by the ability to identify the body fluid origin of single-source and two-fluid admixtures. The discriminatory power (European Caucasians) for all body fluids is 0.957-0.997, with linkage disequilibrium considered. Reciprocal body fluid admixtures (mixture pairs with the same two donors but reversed body fluid types) were used to demonstrate the ability to identify the body fluid source of origin as well as associate the donor of each of the two fluids.

Y-STR mixture deconvolution by single-cell analysis.

Since Y-STR typing only amplifies male Y chromosomal DNA, it can simplify the interpretation of some DNA mixtures that contain female DNA. However, if there are multiple male contributors, mixed Y-STR DNA profiles will often be obtained. Y-STR mixture analysis cases are particularly challenging though as, currently, there are no validated probabilistic genotyping (PG) software solutions commercially available to aid in their interpretation. One approach to fully deconvoluting these challenging mixtures into their individual donors is to conduct single-cell genotyping by isolating individual cells from a mixture prior to conducting DNA typing. In this work, a physical micromanipulation technique involving a tungsten needle and direct PCR with decreased reaction volume and increased cycle number was applied to equimolar 2- and 3-person buccal cell male DNA mixtures and a mock touch DNA case scenario involving the consecutive firing of a handgun by two males. A consensus DNA profiling approach was then utilized to obtain YFiler™ Plus Y-STR haplotypes. Buccal cells were used to optimize and test the direct single-cell subsampling approach, and 2-3 person male buccal cell mixtures were fully deconvoluted into their individual donor Y-STR haplotypes. Single-cell (or agglomerated cell clump) subsampling from the gun's trigger recovered single-source Y-STR profiles from both individuals who fired the gun, the owner, and the other unrelated male. Only the non-owner's DNA was found in the cells recovered from the handle. In summary, direct single-cell subsampling as described represents a potential simple way to analyze and interpret Y-STR mixtures.

Cell Subsampling Recovers Probative DNA Profile Information from Unresolvable/Undetectable Minor Donors in Mixtures.

When a minor DNA component to a binary mixture is present at a weight ratio of approximately 1:50 or less, the presence of this minor donor is undetectable (or barely detectable) by standard mixture deconvolution approaches. In an attempt to retrieve probative minor donor DNA profile information, multiple quintuple cell subsamples were collected from a 1:50 DNA mixture using direct single cell subsampling (DSCS) paired with probabilistic genotyping (PG), the latter validated for use with single or few cells. DSCS employs a simplified micromanipulation technique paired with an enhanced DNA profiling approach, involving direct cell lysis and a sensitive PCR process, to genotype individual cells. Multiple five-cell subsamples were used to interrogate sufficient cells from the mixture such that some of the created 5-cell "mini-mixture" subsamples contained a cell from the minor donor. The latter mini-mixture subsamples, which now comprised weight ratios of 1:4 as opposed to the bulk mixture 1:50, were analyzed with the PG systems STRmixTM and EuroForMix resulting in a significant probative gain of information, (LR ≅ 1011, compared to standard bulk mixture PG methods, LR ≅ 101-102).

Probabilistic genotyping of single cell replicates from complex DNA mixtures recovers higher contributor LRs than standard analysis.

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2 to 6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2-6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (≥1020) from donors that had returned non-probative LRs (<103) by standard methods.

Durability of Clinical and Quality-of-Life Outcomes of Closed-Loop Spinal Cord Stimulation for Chronic Back and Leg Pain: A Secondary Analysis of the Evoke Randomized Clinical Trial.

Importance: Chronic pain is debilitating and profoundly affects health-related quality of life. Spinal cord stimulation (SCS) is a well-established therapy for chronic pain; however, SCS has been limited by the inability to directly measure the elicited neural response, precluding confirmation of neural activation and continuous therapy. A novel SCS system measures the evoked compound action potentials (ECAPs) to produce a real-time physiological closed-loop control system. Objective: To determine whether ECAP-controlled, closed-loop SCS is associated with better outcomes compared with fixed-output, open-loop SCS at 24 months following implant. Design, Setting, and Participants: The Evoke study was a double-blind, randomized, controlled, parallel arm clinical trial with 36 months of follow-up. Participants were enrolled from February 2017 to 2018, and the study was conducted at 13 US investigation sites. SCS candidates with chronic, intractable back and leg pain refractory to conservative therapy, who consented, were screened. Key eligibility criteria included overall, back, and leg pain visual analog scale score of 60 mm or more; Oswestry Disability Index score of 41 to 80; stable pain medications; and no previous SCS. Analysis took place from October 2020 to April 2021. Interventions: ECAP-controlled, closed-loop SCS was compared with fixed-output, open-loop SCS. Main Outcomes and Measures: Reported here are the 24-month outcomes of the trial, which include all randomized patients in the primary and safety analyses. The primary outcome was a reduction of 50% or more in overall back and leg pain assessed at 3 and 12 months (previously published). Results: Of 134 randomized patients, 65 (48.5%) were female and the mean (SD) age was 55.2 (10.6) years. At 24 months, significantly more closed-loop than open-loop patients were responders (≥50% reduction) in overall pain (53 of 67 [79.1%] in the closed-loop group; 36 of 67 [53.7%] in the open-loop group; difference, 25.4% [95% CI, 10.0%-40.8%]; P = .001). There was no difference in safety profiles between groups (difference in rate of study-related adverse events: 6.0 [95% CI, -7.8 to 19.7]). Improvements were also observed in health-related quality of life, physical and emotional functioning, and sleep, in parallel with opioid reduction or elimination. Objective neurophysiological measurements substantiated the clinical outcomes and provided evidence of activation of inhibitory pain mechanisms. Conclusions and Relevance: ECAP-controlled, closed-loop SCS, which elicited a more consistent neural response, was associated with sustained superior pain relief at 24 months, consistent with the 3- and 12-month outcomes.

ECAP-Controlled Closed-Loop Spinal Cord Stimulation Efficacy and Opioid Reduction Over 24-Months: Final Results of the Prospective, Multicenter, Open-Label Avalon Study.

INTRODUCTION: Chronic pain is a major public health concern, as is the associated use of opioid medications, highlighting the importance of alternative treatments, such as spinal cord stimulation (SCS). Here, we present the final 24-month results of the Avalon study, which investigated the use of the first closed-loop SCS system in patients with chronic pain. The system measures the evoked compound action potentials (ECAPs) elicited by each stimulus pulse and drives a feedback loop to maintain the ECAP amplitude near constant. METHODS: Fifty patients were implanted with the Evoke system (Saluda Medical) and followed over 24-months. Pain, quality of life (QOL), function, sleep, and medication use were collected at baseline and each scheduled visit. ECAP amplitudes and programming adjustments were also monitored. RESULTS: At 24 months, responder rates (≥ 50% pain reduction) and high responder rates (≥ 80% pain reduction) for overall pain were 89.5% and 68.4%, respectively, the latter up from 42.2% at 3 months. Significant improvements from baseline were observed in QOL, function, and sleep over the 24 months, including ≥ 80% experiencing a minimally important difference in QOL and > 50% experiencing a clinically significant improvement in sleep. At 24 months, 82.8% of patients with baseline opioid use eliminated or reduced their opioid intake. Over the course of the study, reprogramming need fell to an average of less than once a year. CONCLUSION: Over a 24-month period, the Evoke closed-loop SCS maintained its therapeutic efficacy despite a marked reduction in opioid use and steady decrease in the need for reprogramming.

Forensic transcriptome analysis using massively parallel sequencing.

The application of transcriptome analyses in forensic genetics has experienced tremendous growth and development in the past decade. The earliest studies and main applications were body fluid and tissue identification, using targeted RNA transcripts and a reverse transcription endpoint PCR method. A number of markers have been identified for the forensically most relevant body fluids and tissues and the method has been successfully used in casework. The introduction of Massively Parallel Sequencing (MPS) opened up new perspectives and opportunities to advance the field. Contrary to genomic DNA where two copies of an autosomal DNA segment are present in a cell, abundant RNA species are expressed in high copy numbers. Even whole transcriptome sequencing (RNA-Seq) of forensically relevant body fluids and of postmortem material was shown to be possible. This review gives an overview on forensic transcriptome analyses and applications. The methods cover whole transcriptome as well as targeted MPS approaches. High resolution forensic transcriptome analyses using MPS are being applied to body fluid/ tissue identification, determination of the age of stains and the age of the donor, the estimation of the post-mortem interval and to post mortem death investigations.

Recovery of single source DNA profiles from mixtures by direct single cell subsampling and simplified micromanipulation.

Deconvolution of forensic DNA mixtures into their individual component DNA (geno)types is of great investigative value, though often complex and difficult. Two-person mixtures comprising a major and minor contributor are often easily interpreted although, when the DNA ratio of the two individuals is approximately equal (~1:1), deconvolution and interpretation becomes much more difficult. To address this issue, a physical separation of individual-, two- or three- cell subsamples prior to autosomal STR analysis was performed using a simplified micromanipulation technique paired with a decreased reaction volume and increased cycle number PCR. Using this method, single and multiple buccal epithelial cells were collected from a 1:1 two-person mixture (i.e. from individual 'A' and 'B') and directly amplified, omitting standard DNA extraction and purification steps. Single cell subsamples resulted in partial single-source profiles for both contributors while, in accordance with expectations of a quasi-binomial sampling schema, two- and three-cell subsamples resulted in single source informative partial profiles of individual A and individual B as well as complete consensus profiles, and equally mixed 1:1 (2-cell subsamples) and 2:1 (3-cell subsamples) admixed profiles of individual A and B.This proof-of-concept approach shows promise in permitting the DNA deconvolution of mixed samples where the individual contributors are present in similar amounts that would otherwise be difficult to interpret, resulting in an increase in evidentiary value. The subsampling approach can be readily investigated for DNA casework applications without additional investment in costly, new equipment, requiring only a stereo microscope and a tungsten needle.

Identification of cadaveric liver tissues using thanatotranscriptome biomarkers.

Thanatotranscriptome studies involve the examination of mRNA transcript abundance and gene expression patterns in the internal organs of deceased humans. Postmortem gene expression is indicative of the cellular status of a corpse at the time of death, a portion of which may represent a cascade of molecular events occasioned by death. Specific gene biomarkers identify perceptible transcriptional changes induced by stochastic responses to the cessation of biological functions. Transcriptome analyses of postmortem mRNA from a tissue fragment may determine unique molecular identifiers for specific organs and demonstrate unique patterns of gene expression that can provide essential contextual anatomical information. We evaluated the impact of targeted transcriptome analysis using RNA sequencing to reveal global changes in postmortem gene expression in liver tissues from 27 Italian and United States corpses: 3.5-hour-old to 37-day-old. We found that our single blind study using eight liver tissue-specific gene biomarkers (e.g. AMBP and AHSG) is highly specific, with autopsy-derived organ samples correctly identified as tissues originating from postmortem livers. The results demonstrate that 98-100% of sequencing reads were mapped to these liver biomarkers. Our findings indicate that gene expression signatures of mRNA exposed up to 37 days of autolysis, can be used to validate the putative identity of tissue fragments.

Sustained Long-Term Outcomes With Closed-Loop Spinal Cord Stimulation: 12-Month Results of the Prospective, Multicenter, Open-Label Avalon Study.

BACKGROUND: Spinal cord stimulation (SCS) activates the dorsal column fibers using electrical stimuli. Current SCS systems function in fixed-output mode, delivering the same stimulus regardless of spinal cord (SC) activation. OBJECTIVE: To present long-term outcomes of a novel closed-loop SCS system that aims to maintain the SC activation near a set target level and within a therapeutic window for each patient. SC activation is measured through the evoked compound action potential (ECAP) generated by each stimulus pulse. METHODS: Fifty patients with lower back and/or leg pain who were successfully trialed received a permanent system (Evoke; Saluda Medical, Sydney, Australia). Ratings of pain (visual analog scale), quality of life, function, sleep, and medication use were collected at baseline and at each visit. SC activation levels were reported in summary statistics. The therapeutic window for each individual patient was defined as the range of ECAP amplitudes between sensation threshold and uncomfortably strong stimulation. RESULTS: At 12 mo, the proportion of patients with ≥50% relief was 76.9% (back), 79.3% (leg), and 81.4% (overall), and the proportion with ≥80% pain relief was 56.4% (back), 58.6% (leg), and 53.5% (overall). Patients spent a median of 84.9% of their time with stimulation in their therapeutic window, and 68.8% (22/32) eliminated or reduced their opioid intake. Statistically significant improvements in secondary outcomes were observed. CONCLUSION: The majority of patients experienced more than 80% pain relief with stable SC activation, as measured by ECAP amplitude at 12 mo, providing evidence for the long-term effectiveness of the Evoke closed-loop SCS system.

Zeolite-Templated Carbon as a Stable, High Power Magnesium-Ion Cathode Material.

One strategy to overcome the slow kinetics associated with electrochemical magnesium ion storage is to employ a permanently porous, capacitive cathode material together with magnesium metal as the anode. This strategy has begun to be employed, for example, using framework solids like Prussian blue analogues or porous carbons derived from metal-organic frameworks, but the cycling stability of an ordered, bottom-up synthesized, three-dimensional carbon framework toward magnesiation and demagnesiation in a shuttle-type battery remains unexplored. Zeolite-templated carbons (ZTCs) are a class of ordered porous carbonaceous framework materials with numerous superlative properties relevant to electrochemical energy storage. Herein, we report that ZTCs can serve as high-power cathode materials for magnesium-ion hybrid capacitors (MHCs), exhibiting high specific capacities (e.g., 113 mA h g-1 after 100 cycles) with an average discharge voltage of 1.44 V and exceptional capacity retention (e.g., 76% after 200 cycles). ZTC-based MHCs meet or exceed the gravimetric energy densities of state-of-the-art batteries functioning on the Mg2+ shuttle, while simultaneously displaying far superior rate capabilities (e.g., 834 W kg-1 at 600 mA g-1).

Predicting the origin of stains from whole miRNome massively parallel sequencing data.

In this study, we have screened the six most relevant forensic body fluids / tissues, namely blood, semen, saliva, vaginal secretion, menstrual blood and skin, for miRNAs using a whole miRNome massively parallel sequencing approach. We applied partial least squares (PLS) and linear discriminant analysis (LDA) to predict body fluids based on the expression of the miRNA markers. We estimated the prediction accuracy for models including different subsets of miRNA markers to identify the minimum number of markers needed for sufficient prediction performance. For one selected model consisting of 9 miRNA markers we calculated their importance for prediction of each of the six different body fluid categories.

Recovery of whole mitochondrial genome from compromised samples via multiplex PCR and massively parallel sequencing.

In forensic casework, compromised samples often possess limited or degraded nuclear DNA, rendering mitochondrial DNA a more feasible option for forensic DNA analyses. The emergence of massively parallel sequencing (MPS) has enabled the recovery of extensive sequence information from very low quantities of DNA. We have developed a multiplex PCR method that amplifies the complete mitochondrial genome in a range of forensically relevant samples including single cells, cremated remains, bone, maggot and hairs isolated from dust bunnies. Following library preparation, MPS yields complete or nearly complete mitochondrial genome coverage for all samples. To confirm concordance between sample types and between sequencing platforms, we compared sequencing results from hair and buccal swabs from two references. Low initial DNA input into the multiplex PCR allows for conservation of precious DNA while MPS maximizes recovery of genetic information.

Developmental validation of the ParaDNA® Body Fluid ID System-A rapid multiplex mRNA-profiling system for the forensic identification of body fluids.

Identifying the biological origin of forensic traces can provide crucial evidence to aid criminal investigations. Current forensic practice for the identification of body fluids mostly uses protein-based presumptive tests. Such tests cannot identify all of the forensically relevant fluids and have issues of cross-reactivity. More recently, messenger RNA methods have been developed that have expanded the range of body fluids that can be positively identified. However, these methods are slow and require expert scientists to run the processes and interpret the results. The ParaDNA® Body Fluid ID System has been designed to provide a simple, fast and robust way to identify forensically relevant body fluids in a lab or field-deployable manner. The system can analyse and detect mRNA targets for six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual blood and peripheral blood. Utilising the ParaDNA Sample Collector and instruments, minimal training is required to enable both forensic scientists and non-specialist personnel to analyse biological samples directly, without prior sample processing, in approximately 90 min. The developmental validation studies described here were designed to address requirements of end users, based on the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Body Fluid ID System on a range of mock evidence items. The data collected demonstrate that the Body Fluid ID System can automatically determine the presence of specific body fluid mRNA markers in single-source and mixed samples on multiple substrate types and body fluids could be identified with as little as 0.05ng total RNA and 1µl of the relevant fluid. Results can either be used to support confirmation of source from previously obtained STR DNA profiling results or to improve sample success rates by making better informed evidential submissions.

Single source DNA profile recovery from single cells isolated from skin and fabric from touch DNA mixtures in mock physical assaults.

The ability to obtain DNA profiles from trace biological evidence is routinely demonstrated with so-called 'touch DNA evidence', which is generally perceived to be the result of DNA obtained from shed skin cells transferred from a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such 'blind-swabbing' approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a 'smart analysis' single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male 'assailant' grabbing the wrist, neck or clothing from the female 'victim', or being in transient contact with bedding from the 'victim'). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor 'assailant' DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.

Predicting the origin of stains from next generation sequencing mRNA data.

We used our previously published NGS mRNA approach for body fluid identification to analyse 183 body fluids/tissues, including mock casework samples. The resulting data set was used to build a probabilistic model that predicts the origin of a stain. Our approach uses partial least squares followed by linear discriminant analysis to classify samples into six commonly occurring forensic body fluids. The model differs from the ones previously suggested in that it incorporates quantitative information (NGS read counts) rather than just presence/absence of markers. The suggested approach also allows for visualisation of important markers and their correlation with the different body fluids. We compared our model to previously published methods to show that the inclusion of read count information improves the prediction. Finally, we applied the model to mixed body fluid samples to test its ability to identify the individual components in a mixture.