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Disaster victim identification (DVI) refers to the forensic identification of unknown individuals following a mass disaster event. Human dental structures can contain viable DNA sources when other soft tissues are compromised. However, labor‐intensive sample preparation performed by extensively trained personnel is needed to expose the nuclear material for traditional forensic DNA workflows. With this in mind, we evaluated two simplified sample preparation protocols for processing tooth samples using either a conventional forensic DNA workflow or the Applied Biosystems® RapidHIT™ ID instrument. Briefly, sample sets for both protocols included 10 deciduous teeth that were cleaned prior to either fragmentation with a claw hammer (for RapidHIT™ ID processing) or fine‐powder pulverization with a consumer‐grade coffee grinder (for traditional workflows). The average percentage of expected STR alleles that were detected above analytical threshold for these tooth samples were comparable between methods: RapidHIT™ ID = 99.0% and GlobalFiler™ = 99.8%. Average intralocus heterozygote peak height ratios (PHRs) were comparable: RapidHIT™ ID = 0.80 and GlobalFiler™ = 0.86. Importantly, 9 of 10 samples analyzed via the RapidHIT™ ID required analyst review for flagged artifact peaks and quality issues. Across all profiles, 91% of alleles passed quality metrics for the RapidHIT™ workflow versus 100% for conventional GlobalFiler™ analysis. Collectively, these results suggest that quick, low‐tech tooth sample fragmentation followed by analysis with the RapidHIT™ ID instrument can produce complete STR profiles from aged tooth samples. Future studies should include larger samples sets, more challenging tooth samples, and further simplification of sample preparation to enable field‐forward, on‐scene DVI. [ABSTRACT FROM AUTHOR]

Mass migration and migrant death at the U.S. southern border highlight the disconnectedness of the systems for transnational decedent identifications. Death investigation cases in Texas face delays and barriers at all stages of an investigation. Additionally, fragmentation of DNA databases exacerbate challenges in comparing genetic samples from unidentified human remains (UHR) and families of the missing. We sought to pilot alternative workflows for processing UHR and family reference samples (FRS) for the identification of probable migrant decedents. Primarily using Rapid DNA, but also accredited non-CODIS DNA laboratories, the piloted approaches were conducted in parallel to existing medicolegal workflows under the relevant case jurisdictional guidance. Our data show that Rapid DNA is a valid path for anthropology laboratories to support identification hypotheses and that accredited non-CODIS forensic and genetic laboratories also can support families to identify remains, especially when families reside outside of the United States.

Crime laboratories have been faced with large casework backlogs due to lengthy processing times, limited resources and scientists, and rising crime rates. Evidence related to sexual assault crimes, specifically sexual assault kits (SAKs), heavily contribute to the reported backlogs. Although more sensitive, faster chemistries and automated techniques have been implemented over the years, the traditional STR workflow remains relatively unchanged. Enhanced workflows such as direct PCR and Rapid DNA have the potential to streamline the processing of forensic evidence items including those commonly submitted in SAKs, but the FBI QAS guidelines restrict CODIS‐approved labs from implementing these methods for forensic samples. Recent studies have shown decreased turnaround times and improved or comparable profiling success with both approaches. However, review of the literature shows a lack of in‐depth research comparing traditional DNA workflows to faster and more sensitive direct PCR and/or Rapid DNA approaches for evidentiary samples, especially for SAKs. By providing the forensic science and criminal justice communities with the strengths and limitations of direct PCR and Rapid DNA methods, stakeholders and policy makers may be better informed. [ABSTRACT FROM AUTHOR]

Rapid DNA technology is being utilized for reference profiles worldwide. There is also strong data in the literature to support its use for high‐template DNA sources, the same is not true for low‐template sources, such as touch DNA; this is a requirement before wider implementation to forensic casework is considered. We report on the Rapid HIT Intel cartridge's ability to facilitate successful amplification of touch DNA to obtain profiles from template deposited on items commonly encountered in forensic casework. Eight items were touched in ten replicates— two were tapelifted, three swabbed, and three directly inserted. Significance was observed in the alleles amplified and RFU with respect to sample type. Three samples performed well: cable tie, fabric, and matchstick. As two of these were directly inserted, this should be considered for any sample small enough. Placement of highly absorbent substrates into the cartridge is not advised as it can cause a lysate‐pull error. Heterozygote loci often presented as homozygous (32%–78% loci per profile); this was influenced by substrate type and profile RFU. Loci with larger masses exhibited higher false homozygosity also. Comparison of the donor's profile analyzed was performed against previous datasets analyzing touch DNA through standard workflow, including manual DNA extraction, PCR, and CE separation. These data show that for all substrates, except for a fabric swatch, standard processing is preferential to Rapid HIT analysis. In its current form, rapid DNA technology is not fit for the routine analysis of touch DNA samples in forensic casework. [ABSTRACT FROM AUTHOR]

In 2019 and 2020, disaster victim identification (DVI) simulations were conducted at the Australian Facility for Taphonomic Experimental Research. Whole and fragmented cadavers were positioned to replicate a building collapse scenario and left to decompose for up to 4 weeks. This study evaluated the utility of the ANDE™ 6C Rapid DNA System and the RapidHITTM ID System for DVI in the field and mortuary. Applying post-mortem nail and tissue biopsy samples showed promise, with the added benefit of minimally invasive collection procedures and limited preparation requirements. The preferred platform will depend on a number of factors, including its intended use and operating environment. [ABSTRACT FROM AUTHOR]

Short tandem repeat (STR) profiling is one of the mostly used systems for forensic applications. In certain circumstances, STR profiling is time-consuming and costly, which potentially leads to delays in criminal investigations. LGC (Laboratory of the Government Chemist, UK) Forensics has developed a robust STR profiling platform called the ParaDNA® Intelligence Test System which can provide early tactical intelligence and aid investigators in making informed decisions on sample prioritization for detection. Here, we validated the ParaDNA intelligence test for its application in forensic cases using a range of mock evidence items following guidelines set by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Specifically, we tested the sensitivity and accuracy of the ParaDNA intelligence test, as well as the success rates for detecting mock samples and for use in case scenarios. Our findings demonstrate that the ParaDNA intelligence test generates useful DNA profiles, especially for samples such as blood, saliva, and semen that contain ample DNA, indicating the benefits of including ParaDNA as a prior step in forensic STR profiling pipelines.

Rapid DNA platforms are fully automated systems capable of processing DNA from biological samples and interpreting the results in approximately 90 minutes with minimal human intervention. With a greater reliance on the system than on the analyst, validation data are especially needed to define the performance and limitations of commercially available Rapid DNA systems. Thus, validation studies of a Rapid DNA workflow consisting of the Applied Biosystems RapidHIT ID Instrument and RapidLINK software with a focus on the ACE GlobalFiler Express Sample Cartridge and reference buccal swabs were performed in accordance with Scientific Working Group on DNA Analysis Methods Validation Guidelines. These validation studies included assessments of sensitivity, contamination, concordance, reproducibility and repeatability, stability, inhibition, mixtures, sample reprocessing, precision, and first-pass success rate. Overall, the current Applied Biosystems RapidHIT ID Instrument with the ACE GlobalFiler Express sample cartridge was found to be a reliable tool for generation of STR profiles from reference-type buccal swabs. [ABSTRACT FROM AUTHOR]

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In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples.

Evaluating the short tandem repeat (STR) in the field is important for the timely identification of a suspect. Several lines showed that the RapidHIT® ID system is reliable for DNA genotyping with buccal swabs and naked DNA. However, the application of this approach with blood samples has been poorly investigated. Because blood samples are among the most common forensic samples in our laboratory, further studies should be conducted. Here, we assessed the analytical performance of 19 STR loci with a newly developed RapidINTEL (RI) Sample Cartridge Kit by using the blood samples with known genotypes. Several commonly used substrates were included in the sensitivity study, and FTA cards proved to be the most promising sample carrier for blood storage and later identification. There was superior sensitivity and specificity with a 100% concordance rate for 0.5 μL of blood or 7 ng of genomic DNA. The performance for blood samples was comparable with that for the standard protocol. High success rate (90.57%) and high-concordance (100%) genotyping were automatically achieved over a wide range of operating conditions except for TH01. No contamination was observed throughout the study. Hematin, indigo, and humic acid had limited influence on the instrument system, while urea and melanin dramatically affected the genotyping results. Generally, the newly developed RI sample cartridge provided an alternative method for the STR genotyping of single-source blood samples over a wide range of operating conditions. [ABSTRACT FROM AUTHOR]

The RapidHIT™ ID (RHID) system was evaluated for its suitability in processing a single hair root to obtain informative DNA profiles. Hair samples were assessed for nuclear DNA prior to DNA analysis using Diamond™ Nucleic Acid Dye (DD) and real-time Extended Depth of Field (EDF) imaging to visualise and count nuclei if present. Hairs were viewed under an Optico N300F LED Fluorescent Microscope and imaged using a MIchrome 5 Pro camera. Hair roots were processed through both the ACE GlobalFiler™ Express sample cartridge and the RapidINTEL™ sample cartridge. A total of 44 hairs including shed hairs (9) and plucked hairs (35) from 8 donors were evaluated in this study. The processing of hairs using the RHID system required the modification of a standard swab that allowed for hairs to be easily collected and placed into the cartridge but also allowed for the re-collection of hair roots post RHID analysis (for potential standard DNA workflow). 90% of plucked hairs with a high nuclei count (>100) resulted in a high partial or full DNA profile, with the remaining 10% resulting in a low partial profile. 44% of shed hairs resulted in a low partial profile, with the remaining hairs resulting in a null profile. This study demonstrated that the RHID system could successfully obtain a DNA profile from a single hair root with nuclei present post-DD staining. According to these results, it is suggested that when dealing with hairs containing fewer than 50 nuclei, using the RapidINTEL™ cartridge can enhance allele recovery. • A modified swab was required for the analysis of hair roots using the RapidHIT™ ID system. • Hair roots were assessed for the presence of DNA using Diamond™ Nucleic Acid Dye. • Full DNA profiles were obtained from stained hairs with visible nuclei using the RapidHIT™ ID system. • 90% of plucked hair samples (>100 nuclei count) resulted in a high partial or full DNA profile using the RapidHIT™ ID system. [ABSTRACT FROM AUTHOR]

The extraction of DNA is a fairly standard procedure routinely undertaken in biological academic and research settings. In a forensic DNA context, DNA extraction is the crucial first step toward the generation of DNA profiles used to support convictions or exonerations in the criminal justice system. However, it is often complicated by the additional challenge of yielding sufficient quantities of clean DNA that is free from environmental inhibitors, from a wide spectrum of sample types which may contain limited quantities of biological material. While improvements in methods and technology over the decades have sped up and simplified the process of DNA extraction, several issues remain to be fully addressed—such as the incomplete separation of spermic DNA from sperm‐epithelial cell mixtures, increasing demands for ever‐faster DNA results, or even on‐scene DNA processing, and processing of difficult samples such as skeletal remains or samples contaminated by Chemical, Biological, or Radiological (CBR) agents. These challenges are often compounded by high work volumes and limited quantities of starting material. As such, this review focuses on recent trends and developments in four areas of forensic DNA extraction: (a) analysis of sexual assault evidence which often represents a significant portion of a forensic DNA laboratory's casework, (b) use of portable rapid DNA instruments which has gained much traction among law enforcement in recent times, (c) DNA extraction from difficult and CBR‐contaminated samples which, albeit rare, would be essential in a DNA laboratory's toolbox in the event of an incident, and (d) bypassing DNA extraction altogether. This article is categorized under:Forensic Biology > Forensic DNA Technologies [ABSTRACT FROM AUTHOR]

Short tandem repeat (STR) profiling is one of the mostly used systems for forensic applications. In certain circumstances, STR profiling is time-consuming and costly, which potentially leads to delays in criminal investigations. LGC (Laboratory of the Government Chemist, UK) Forensics has developed a robust STR profiling platform called the ParaDNA® Intelligence Test System which can provide early tactical intelligence and aid investigators in making informed decisions on sample prioritization for detection. Here, we validated the ParaDNA intelligence test for its application in forensic cases using a range of mock evidence items following guidelines set by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Specifically, we tested the sensitivity and accuracy of the ParaDNA intelligence test, as well as the success rates for detecting mock samples and for use in case scenarios. Our findings demonstrate that the ParaDNA intelligence test generates useful DNA profiles, especially for samples such as blood, saliva, and semen that contain ample DNA, indicating the benefits of including ParaDNA as a prior step in forensic STR profiling pipelines. [ABSTRACT FROM AUTHOR]

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8 h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2 h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10 ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories., (© 2024 Wiley‐VCH GmbH.)

Three commercially available integrated rapid DNA instruments were tested as a part of a rapid DNA maturity assessment in July of 2018. The assessment was conducted with sets of blinded single‐source reference samples provided to participants for testing on the individual rapid platforms within their laboratories. The data were returned to the National Institute of Standards and Technology (NIST) for review and analysis. Both FBI‐defined automated review (Rapid DNA Analysis) and manual review (Modified Rapid DNA Analysis) of the datasets were conducted to assess the success of genotyping the 20 Combined DNA Index System (CODIS) core STR loci and full profiles generated by the instruments. Genotype results from the multiple platforms, participating laboratories, and STR typing chemistries were combined into a single analysis. The Rapid DNA Analysis resulted in a success rate of 80% for full profiles (85% for the 20 CODIS core loci) with automated analysis. Modified Rapid DNA Analysis resulted in a success rate of 90% for both the CODIS 20 core loci and full profiles (all attempted loci per chemistry). An analysis of the peak height ratios demonstrated that 95% of all heterozygous alleles were above 59% heterozygote balance. For base‐pair sizing precision, the precision was below the standard 0.5 bp deviation for both the ANDE 6C System and the RapidHIT 200. [ABSTRACT FROM AUTHOR]

Rapid DNA profiling of casework samples is a powerful tool that can support law enforcement agencies in the quick apprehension of perpetrators before they re-offend or escape the jurisdiction. This present study evaluated the feasibility of direct PCR amplification, using the microFLOQ™ Direct swab, for generating DNA profiles (from bloodstains) within 3 h. The swab tip is coated with nylon fibers pre-treated with cell lysing agent, which allows for the direct PCR amplification of collected samples without DNA extraction and quantification, thereby shortening the time required to obtain a DNA profile. Samples collected were directly amplified using GlobalFiler™ PCR Amplification Kit with and without the presence of a PCR additive. Addition of the PCR additive enhanced the peak heights of DNA profiles by approximately 2 fold. Hence, an additive could improve results obtained in the absence of a DNA purification step, especially since casework samples may contain PCR inhibitors. Subsequently, these swabs, amplified using the GlobalFiler™ PCR Amplification Kit with PCR additive, were evaluated on common substrates encountered in routine casework samples submitted with bloodstains, such as denim jeans, knife blade, tissue paper, leather belt, shirt, and blood swabs. The minimum peak heights observed were generally above the analytical and stochastic thresholds established by the laboratory. Finally, the microFLOQ™ Direct swab workflow was compared to the laboratory's standard workflow of DNA profiling comprising of conventional processing steps such as extraction using the DNA-IQ™ chemistry on Maxwell® 16, followed by quantification, amplification and capillary electrophoresis. The average peak heights of the DNA profiles generated by direct PCR amplification were similar or exceeded those generated using the standard workflow. This study clearly demonstrates that direct PCR amplification using microFLOQ™ Direct swab can be used in a rapid workflow to obtain DNA profiles from casework samples. [ABSTRACT FROM AUTHOR]

A new rapid DNA solution, the RapidHIT®ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low‐throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120 min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost‐saving, efficient design to generate a validated RapidHIT®ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120 min. [ABSTRACT FROM AUTHOR]

A new rapid DNA solution, the RapidHIT®ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low‐throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120 min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost‐saving, efficient design to generate a validated RapidHIT®ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120 min. [ABSTRACT FROM AUTHOR]

Rapid DNA technology has the ability to provide DNA analysis results in real-time. The purpose of this study was to assess the potential investigative value that could be gained by using a rapid DNA workflow in Australia, for reference DNA samples. A survey of police charging stations and DNA analysis laboratories in five Australian jurisdictions identified that a rapid DNA analysis workflow could have impacted the decision to release a person of interest from custody in 0.6% cases. This equates to approximately 480 people per year across Australia who could be held in custody because of DNA results obtained in real time. The survey also identified the extent of a sample backlog in one jurisdiction and highlighted practices in place which limited the number of reference DNA samples collected, thereby reducing the potential impact of the DNA database. It is anticipated that while the cost of rapid DNA technology remains significantly greater than current laboratory processing, the uptake of the technology in Australia will be limited. However, as costs reduce, rapid DNA instruments could potentially become a viable option for the future of forensic science in Australia. [ABSTRACT FROM AUTHOR]

Abstract Mobile Rapid DNA technology is close to being incorporated into crime scene investigations, with the potential to identify a perpetrator within hours. However, the use of these techniques entails the risk of losing the sample and potential evidence, because the device not only consumes the inserted sample, it is also is less sensitive than traditional technologies used in forensic laboratories. Scene of Crime Officers (SoCOs) therefore will face a 'time/success rate trade-off' issue when making a decision to apply this technology. In this study we designed and experimentally tested a Decision Support System (DSS) for the use of Rapid DNA technologies based on Rational Decision Theory (RDT). In a vignette study, where SoCOs had to decide on the use of a Rapid DNA analysis device, participating SoCOs were assigned to either the control group (making decisions under standard conditions), the Success Rate (SR) group (making decisions with additional information on DNA success rates of traces), or the DSS group (making decisions supported by introduction to RDT, including information on DNA success rates of traces). This study provides positive evidence that a systematic approach for decision-making on using Rapid DNA analysis assists SoCOs in the decision to use the rapid device. The results demonstrated that participants using a DSS made different and more transparent decisions on the use of Rapid DNA analysis when different case characteristics were explicitly considered. In the DSS group the decision to apply Rapid DNA analysis was influenced by the factors "time pressure" and "trace characteristics" like DNA success rates. In the SR group, the decisions depended solely on the trace characteristics and in the control group the decisions did not show any systematic differences on crime type or trace characteristic. Guiding complex decisions on the use of Rapid DNA analyses with a DSS could be an important step towards the use of these devices at the crime scene. Highlights • A Decision Support System (DSS) was designed for using a mobile Rapid DNA device. • With DSS guidance different and more thoughtful Rapid DNA decisions were made. • DSS showed a significant effect on the factors 'time pressure' and 'trace type'. • Knowledge on DNA success rates is essential to support decisions for Rapid DNA device use. [ABSTRACT FROM AUTHOR]

Mass migration and migrant death at the U.S. southern border highlight the disconnectedness of the systems for transnational decedent identifications. Death investigation cases in Texas face delays and barriers at all stages of an investigation. Additionally, fragmentation of DNA databases exacerbate challenges in comparing genetic samples from unidentified human remains (UHR) and families of the missing. We sought to pilot alternative workflows for processing UHR and family reference samples (FRS) for the identification of probable migrant decedents. Primarily using Rapid DNA, but also accredited non-CODIS DNA laboratories, the piloted approaches were conducted in parallel to existing medicolegal workflows under the relevant case jurisdictional guidance. Our data show that Rapid DNA is a valid path for anthropology laboratories to support identification hypotheses and that accredited non-CODIS forensic and genetic laboratories also can support families to identify remains, especially when families reside outside of the United States., Competing Interests: Jessi Brown, Eugene Tan and Richard Selden are employees of and Isabella-Marie Selden was a student intern at ANDE Corporation., (© 2023 The Authors. Published by Elsevier B.V.)

The RapidHIT™ 200 System is a fully automated sample-to-DNA profile system designed to produce high quality DNA profiles within 2h. The use of RapidHIT™ 200 System within the United Kingdom Criminal Justice System (UKCJS) has required extensive development and validation of methods with a focus on AmpFℓSTR® NGMSElect™ Express PCR kit to comply with specific regulations for loading to the UK National DNA Database (NDNAD). These studies have been carried out using single source reference samples to simulate live reference samples taken from arrestees and victims for elimination. The studies have shown that the system is capable of generating high quality profile and has achieved the accreditations necessary to load to the NDNAD; a first for the UK. [ABSTRACT FROM AUTHOR]

The turn-around time of urgent crime scene DNA samples is often far longer than desired by law enforcement. Crime scene DNA sample processing involves both complex and routine processing steps. Simplification and integration of the routine steps would dramatically improve turn-around time and reduce the risk of operator contamination. Routine DNA extraction and quantitation is readily available. However, PCR amplification and electrophoretic analysis remain largely manual. Rapid DNA Analysis is a hands-free “swab in – profile out” process which consists of automated DNA extraction, amplification, separation, detection, and allele calling without human intervention. RapidHIT ® 200 and RapidHIT ID are rapid DNA systems developed by IntegenX (Pleasanton, CA) and validated for the use of buccal swabs. A new generation of the RapidHIT sample cartridge for RapidHIT ID has been designed and tested which allows the loading of extracted and quantified DNA. RapidHIT EXT sample cartridge allows a user to generate a forensic DNA profile from less than 250 pg of extracted and quantified DNA in less than 90 min with less than one-minute hands-on time. Once the sample is loaded in the RapidHIT EXT sample cartridge, a DNA profile is produced after amplification, detection and automated data analysis. We report on sensitivity, reproducibility, concordance, DNA mixtures and carryover for EXT sample cartridges pre-loaded with GlobalFiler ® Express and AmpFLSTR ® NGM SElect™ Express, (Thermo Fisher Scientific, Waltham, MA) STR chemistries. Purified and quantified DNA from mock crime scene samples were used to demonstrate the utility of these cartridges in an established forensic laboratory. [ABSTRACT FROM AUTHOR]

Traditionally, forensic DNA analysis has required highly skilled forensic geneticists in a dedicated laboratory to generate short tandem repeat (STR) profiles. STR profiles are routinely used either to associate or exclude potential donors of forensic biological evidence. The typing of forensic reference samples has become more demanding, especially with the requirement in some jurisdictions to DNA profile arrestees. The Rapid DNA (RDNA) platform, the RapidHIT ® ID (IntegenX ® , Pleasanton, CA), is a fully automated system capable of processing reference samples in approximately 90 min with minimal human intervention. Thus, the RapidHIT ID instrument can be deployed to non-laboratory environments (e.g., booking stations) and run by trained atypical personnel such as law enforcement. In order to implement the RapidHIT ID platform, validation studies are needed to define the performance and limitations of the system. Internal validation studies were undertaken with four early-production RapidHIT ID units. Reliable and concordant STR profiles were obtained from reference buccal swabs. Throughout the study, no contamination was observed. The overall first-pass success rate with an “expert-like system” was 72%, which is comparable to another current RDNA platform commercially available. The system’s second-pass success rate (involving manual interpretation on first-pass inconclusive results) increased to 90%. Inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect typing by the instrument system; however, substrate (i.e., swab type) did impact typing success. Additionally, one desirable feature not available with other Rapid systems is that in the event of a system failed run, a swab can be recovered and subsequently re-analyzed in a new sample cartridge. Therefore, rarely should additional sampling or swab consumption be necessary. The RapidHIT ID system is a robust and reliable tool capable of generating complete STR profiles within the forensic DNA typing laboratory or with proper training in decentralized environments by non-laboratory personnel. [ABSTRACT FROM AUTHOR]

Rapid DNA platforms are fully integrated systems capable of producing and analyzing short tandem repeat (STR) profiles from reference sample buccal swabs in less than two hours. The technology requires minimal user interaction and experience making it possible for high quality profiles to be generated outside an accredited laboratory. The automated production of point of collection reference STR profiles could eliminate the time delay for shipment and analysis of arrestee samples at centralized laboratories. Furthermore, point of collection analysis would allow searching against profiles from unsolved crimes during the normal booking process once the infrastructure to immediately search the Combined DNA Index System (CODIS) database from the booking station is established. The DNAscan/ANDE™ Rapid DNA Analysis™ System developed by Network Biosystems was evaluated for robustness and reliability in the production of high quality reference STR profiles for database enrollment and searching applications. A total of 193 reference samples were assessed for concordance of the CODIS 13 loci. Studies to evaluate contamination, reproducibility, precision, stutter, peak height ratio, noise and sensitivity were also performed. The system proved to be robust, consistent and dependable. Results indicated an overall success rate of 75% for the 13 CODIS core loci and more importantly no incorrect calls were identified. The DNAscan/ANDE™ could be confidently used without human interaction in both laboratory and non-laboratory settings to generate reference profiles. [ABSTRACT FROM AUTHOR]

Recent advancements in DNA analysis have seen the emergence of Rapid DNA technology. This technology effectively combines the laboratory analytical processes involved in DNA analysis into one step: sample in to result out. This is achieved in a much shorter timeframe than standard DNA analysis; typically in about 1.5 hr. Rapid DNA technology has the potential to be used outside of the laboratory environment and, in some cases, be truly portable. There are numerous legislative and policy considerations by management for the implementation of this type of technology. This will include implications, applications, and limitations of these types of instruments, including validation and policy issues, legal questions, integration to databases, and accreditation requirements. [ABSTRACT FROM AUTHOR]

Equivalent amounts of compromised bones were used to directly compare STR success of conventional and Rapid DNA methods. Conventional DNA extraction methods, including manual full demineralization and semi-automated PrepFiler BTA/ AutoMate Express (ThermoFisher Scientific), provided insights regarding the DNA quantity and extent of degradation of each compromised bone analyzed with ANDE 6C (ANDE Corp) and RapidHIT ID (ThermoFisher Scientific) Rapid systems. Full demineralization provided higher DNA yields than extraction with the AutoMate Express for quality control (QC) and environmentally challenged bones. The degradation indices ranged from ∼1.8 to 73. Both demineralization and AutoMate Express extracts benefited from additional clean-up with NucleoSpin XS devices, which usually resulted in more alleles being detected than without further clean-up. Complete "CODIS 20″ profiles could be obtained with bone QC1 with all methods. However, among the 14 compromised bones with low DNA content, complete CODIS 20 profiles were detected for 7, 4, and 0 bones analyzed with demineralization, AutoMate Express and ANDE methods, respectively. The RapidHIT ID was the least sensitive method, providing the fewest detectable alleles for the bones tested. Whereas extracted DNA of approximately 0.1 ng can yield complete GlobalFiler STR profiles, at least 30 ng was required for complete FlexPlex 27 profiles using the ANDE 6C Rapid DNA system. In addition to being less sensitive than conventional methods, the tested Rapid DNA approaches were less predictable when attempting to improve STR success and proved to be less reliable in genotyping accuracy. • Conventional methods were more successful than Rapid methods for STR typing of compromised bones. • Erroneous and false homozygous genotypes were detected among bones run on the ANDE system. • Modified rapid analysis was required for genotyping of compromised bones with ANDE and RHID systems. • Optimization of bone processing time and quantity on the ANDE system did not improve genotyping success. [ABSTRACT FROM AUTHOR]

Forensic laboratories are universally acknowledged as being overburdened, underfunded, and in need of improved analytical methods to expedite investigations, decrease the costs associated with nucleic acid (NA) analysis, and perform human identification (HID) at the point of need (e.g., crime scene, booking station, etc.). In response, numerous research and development (R&D) efforts have resulted in microfluidic tools that automate portions of the forensic genetic workflow, including DNA extraction, amplification, and short tandem repeat (STR) typing. By the early 2000 s, reports from the National Institute of Justice (NIJ) anticipated that microfluidic 'swab-in-profile-out' systems would be available for use at the crime scene by 2015 and the FBI's 2010 'Rapid DNA' Initiative, approved by Congress in 2017, directed this effort by guiding the development and implementation of maturing systems. At present, few fully-automated microfluidic DNA technologies are commercially available for forensic HID and their adoption by agencies interested in identification has been limited. In practice, the integration of complex laboratory processes to produce one autonomous unit, along with the highly variable nature of forensic input samples, resulted in systems that are more expensive per sample and not comparable to gold-standard identification methods in terms of sensitivity, reproducibility, and multiplex capability. This Review and Perspective provides insight into the contributing factors to this outcome; namely, we focus on the complications associated with the tremendous undertaking that is developing a sample-in-answer-out platform for HID. For context, we also describe the intricate forensic landscape that contributes to a nuanced marketplace, not easily distilled down to cases of simple supply and demand. Moving forward and considering the trade-offs associated with developing methods to compete, sometimes directly, with conventional ones, we recommend a focus shift for microfluidics developers toward the creation of innovative solutions for emerging applications in the field to increase the bandwidth of the forensic investigative toolkit. Likewise, we urge case working personnel to reframe how they conceptualize the currently available Rapid DNA tools; rather than comparing these microfluidic methods to gold-standard procedures, take advantage of their rapid and integrated modes for those situations requiring expedited identifications in an informed manner. • Tools that enable forensic human identification at the point-of-need have the potential to expedite investigations. • Forensic microfluidic technologies have unique advantages and practical limitations. • The research and development of automated methods to mimic forensic workflows have resulted in few commercial systems. • The integration of existing microfluidic platforms is discussed and opportunity zones for future innovation are proposed. [ABSTRACT FROM AUTHOR]

The Proactive Crime Scene Response is a technique utilizing targeted forensic analytical results to guide criminal investigations in real time. Analytical value of evidence maximized by forensic laboratories is directly related to the recognition, documentation, collection, and preservation of evidentiary items located at the crime scene. Improved education, coordination and communication between the crime scene investigators and forensic scientist experts creates a seamless analytical process flow, enabling greater focus on high value evidence with decreased response time and greater impact on investigational direction. Real time data from focused forensic analyses and use of databases provides primary investigative leads, with suspect identities, whereabouts at the time of crime commission, links to other crimes and other critical collaborative crime solving information. Case examples highlighting successful application of various aspects of this model will be provided, with recommendations for implementation including Rapid DNA and supporting business cases., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The author collaborates with Dr. Igor Lednev, SUNY Albany and SupreMEtric. The author is the Chair of the National Institute of Standards and Technology (NIST) Forensic Science Standards Board (FSSB) of the Organization of Scientific Area Committees (OSAC) for Forensic Science., (© 2023 The Author(s).)

The RapidHIT™ 200 Human Identification System and RapidHIT GlobalFiler ® Express kit were evaluated and validated for use with single-source reference samples. It was of primary interest to evaluate the system for its efficacy as an expert system and to estimate a first pass success rate, as well as to identify the technical variables impacting that result. While results indicated that this instrument/kit combination can be used to accurately type single-source buccal samples, substantial variability in sensitivity and intra-color balance were observed, as were multiple artifacts, requiring extensive manual editing of the profiles. Artifacts included dye “blobs” and spectral overlap (pull-up) peaks that often originated from relatively low intensity allele peaks. Reduced intra-color balance, in combination with low sensitivity, occasionally resulted in instances of allelic dropout. Overall, 50% of the buccal samples analyzed in this study would have been successfully typed to give full GlobalFiler ® profiles without the need for manual review and editing. [ABSTRACT FROM AUTHOR]