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The use of DNA in criminal cases has rapidly grown in recent years and forensic labs are confronted with more samples to analyze, track and store. At the same time, many states have passed legislation to include arrestee DNA samples for analysis, adding to backlogs. Fortunately, new tools for automating DNA sample processing are helping labs increase throughput and reliability, improve chain of custody and reduce errors.
The manual workflow for preparing casework DNA samples is tedious and comprises multiple steps that include lysis, DNA extraction and purification, quantification and normalization, Short Tandem Repeat (STR) plate setup and STR amplification. Finally, capillary electrophoresis (CE) plate setup and STR profile analysis are usually carried out on a separate robotic system.
Robotic liquid handling instruments can automate all or part of this process. A single system can manage the core forensic DNA sample processing methods like extraction, quantification and dilution STR setup. There are additional robotic systems for other aspects of the casework DNA process, such as dedicated lysis or CE sequencing setup.
Many casework samples require a prior lysis step, which can be a major bottleneck. A wide variety of substrates must be handled, using spin baskets or filter columns, to generate cleared lysates for downstream extraction. Until recently this was a tedious, error-prone process requiring manual pipetting, which made sample tracking difficult. Dedicated workstations can fully automate the lysis step, and lysate recovery can now be incorporated into a DNA processing workflow. If desired, lysis and extraction can be combined into a single instrument.
While one robotic system can handle both lysis and extraction, many large labs desire a dedicated DNA extraction system. This system will contain the hardware and software to automate DNA purification using magnetic bead methods. An integrated heater/shaker with adapters for extraction kit processing plate is needed. Vacuum filtration hardware can also be integrated. For optimal flexibility, the robotic extraction module options should support a variety of purification methods from different vendors as well as allow tube or plate input and output formats.
Quantification, Normalization and STR Setup
If a forensic lab has a DNA extraction system in place, management may consider adding a dedicated system for post-purification PCR setup. This system would automate quantification, normalization/dilution and STR setup. Quantification is carried out using real-time quantitative PCR (qPCR) or plate reader-based optical methods, while normalization can be achieved via imported qPCR files or plate reader DNA concentration files. A number of STR assay kits are available for the amplification step.
Labs may opt to purchase an application-specific system customized for human identification. Designed to streamline the different processes used by most casework DNA forensics laboratories, a system like this can be configured as a dedicated extraction solution, dedicated PCR solution, or a complete solution for both extraction and PCR. Such a system ensures a high level of sample traceability, quality DNA methods, robustness, and can be integrated with common sample management systems and is compatible with most vendors’ forensic reagent kits.
Automating the Entire Pre-PCR Process
Automation brings with it tools that support sophisticated sample ID tracking, tip counting and error handling to maintain sample traceability throughout the entire DNA sample preparation and pre-PCR analysis process. Labs can choose a platform that automates the entire DNA workflow on a single instrument, processing up to 96 samples.
After STR amplification, a separate robotic platform should be used to carry out CE plate setup. This follows guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Although sample preparation for CE can be carried out with large, complex systems, an affordable, an entry-level 2- or 4-probe pipettor is an easy-to-implement alternative.
In many database labs, punch-card samples are analyzed using direct amplification methods. PCR master mix is added to a punch in a PCR plate well, amplified and prepared for CE analysis. Current instrumentation integrates punching and liquid handling on a single platform with a workflow that seamlessly carries out fully automated punching and master mix addition for blood- and saliva-stained card samples.
Automating Sample Management and Storage
While sample preparation and analysis are principal tasks in a forensic DNA lab, the management, tracking and storage of those samples can introduce problems if not carried out properly. Manual storage in freezers has been the de facto standard in forensic labs, but rising sample numbers have brought to light the limitations of even the most organized manual systems. Finding samples becomes increasingly difficult as the number of samples and freezers increases. Cataloging samples within one or more freezers is time-consuming and inherently challenging and these records are prone to human errors.
Sample degradation is a serious, yet often overlooked limitation of manual freezers. As samples are retrieved and re-stored, freezer doors are opened, creating temperature variations that can compromise sample integrity. Improper placement of sample racks in the freezer can also lead to significant variation in the internal temperature.
Security and access controls are also major factors to consider in sample management and storage. Although most laboratories have some security measures such as lock boxes or restricted access, these basic precautions may not suffice for those labs that handle large numbers of samples. Deficiencies in sample handling protocols and lack of an audit trail could prove to be the weak link in a case that relies on forensic evidence.
Automated sample management systems can overcome many of these issues by providing end-to-end handling and tracking across all aspects of sample management. Multi-featured systems keep track of samples, control access and provide storage for a range of conditions, from ambient, low-humidity settings for FTA cards, to -20 ˚C for biological samples. These systems typically include sophisticated robotics that can handle a variety of sample plates or tubes and can even interface with a lab’s sample processing modules. A modular system can be scaled to both the capacity requirements and footprint constraints of individual labs. To fully automate the sample management process, benchtop units carry out capping and decapping functions for samples in microtubes and can efficiently process a full rack of 48 or 96 tubes. There is an automated decapping device on the market with an integrated tube reader that can rapidly read both 1-D and 2-D barcodes, so the samples can be tracked from pre- to post-processing, providing chain of custody throughout the workflow. Additionally, sample security is ensured by only uncapping one row of tubes at a time, avoiding the risk of cross-contamination.
Automated sample management and storage systems come with a variety of specifications and configurations, and the ideal system for a forensic laboratory will depend on the type and number of samples being managed. Chemical samples, which include post-mortem fluids and blood alcohol samples, may be stored at 4 ˚C or –20 ˚C, while biological samples may be stored either at –20 ˚C, –80 ˚C or -150 °C depending on the application.
A low-capacity system storing up to 800 plates or 60,000 microtubes has a small footprint and easily fits into an existing laboratory. High-capacity systems storing up to millions of samples are obviously larger but still take up less space than manual freezers for the same number of samples. Recent innovations in tube racks also maximize storage space.
An automated sample management system goes a long way to ensure sample integrity. Temperature is monitored throughout the system, and the user can be alerted to any changes. The robotics can cherry-pick desired samples, eliminating the need to open and close a freezer door and reducing freeze-thaw cycles.
Sample tracking is carried out by 1-D or 2-D barcode readers, and integrated software tracks samples through entry, retrieval and long-term storage. The manager can set up varying degrees of user access privileges, designating access to a specific sample or sets of samples. A complete audit trail can easily be produced with information on who retrieved the sample, when, how many times, and for how long. Similarly, storage history reports can be generated that detail temperature profile, freeze-thaw cycles and sample aging. This documentation can be invaluable in court testimony.
For laboratories that require medium- to high-capacity storage, there are ultra-low-temperature sample management systems with scalable capacity to store from 100,000 to over 10 million samples at temperatures down to –80 ˚C.
Automated Systems Deliver Multiple Benefits
Overall, more forensic labs are looking for turnkey automation solutions to quickly implement and validate in their workflows. Chain of custody, which is possible with fully automated solutions, is more important than ever. Additionally, due to the need to store DNA samples long-term for future reanalysis, advanced management and storage systems are in higher demand.
Automated sample preparation, handling and storage systems improve efficiency, save time, reduce human error and improve documentation, enabling forensic scientists to focus on analysis and interpretation rather than process tasks.
Lois C. Tack, Ph.D., is a Forensic Scientific Leader at Hamilton Robotics
Laurent Baron is a Forensics Market Segment Manager at Hamilton Storage