Automation can increase a forensics lab's sample processing capacity, but it can also add to the complexity of system verification and validation.
It’s no secret that genomic DNA has become a key evidentiary component to many criminal investigations. Increasingly sophisticated sample processing and analytical methods have enhanced the integrity and value of DNA evidence in forensic casework, and the development of large forensic DNA databases has provided an effective mechanism for positive identification of suspects. Despite these advances, it’s also no secret that the forensic community faces many challenges. New legislation has led to an explosion in the number of convicted offender samples earmarked for database inclusion. This, coupled with an enhanced ability to extract and successfully analyze DNA evidence from “cold cases,” has led to a significant backlog with literally tens of thousands of samples waiting to be processed and analyzed.
As a result, many forensic labs are looking to automation to dramatically increase their sample processing capacity. Robotic liquid handling systems, applied to processes such as DNA extraction, quantification, normalization, PCR setup, and post-reaction cleanup can streamline repetitive tasks, improve reproducibility, reduce sample cross-contamination, and allow forensic scientists to spend more time in analysis and data review rather than sample processing. However, automation also brings new challenges that can add to the complexity of system verification and validation, especially for laboratories unfamiliar with robotic systems or those lacking time and/or personnel resources.
Conforming to guidelines
Under the FBI and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines covering forensic labs, both the automated methods and the instruments themselves must be validated prior to use in forensic casework. In addition to writing the robotic protocols, laboratories must verify that the automated platform delivers acceptable pipetting performance and will execute the necessary protocol steps correctly and in the proper sequence.
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Figure 1: TADM software screenshot showing high/low tolerance bands and pressure curves of successfully pipetted extracted DNA samples. (Source: Hamilton Robotics) |
Beyond verification, a formal validation is also required and represents a more comprehensive audit of the automated system and chemistry, encompassing additional criteria such as NIST concordance, stutter, chain-of-custody (sample tracking), sensitivity, and mixture studies. The skills required to conduct this method scripting and validation lean more toward bioengineering than biology, and the tasks can be difficult for forensic lab personnel. In a process that can take as long as two years, conducting verification and validation in-house diverts analysts from important casework and adds to database backlog. Additionally, some laboratory automation companies have overpromised and underdelivered, leaving forensics customers with an instrument or process that cannot readily be validated and, therefore, can no longer be implemented to increase the lab’s efficiency.
To address these validation hurdles and help forensic labs apply automation more quickly and efficiently, Hamilton Robotics and Sorenson Forensics entered into a strategic collaboration last year. Working closely with leading forensic DNA chemistry providers, the joint venture has resulted in hardware, software, and documentation packages that include fully optimized, prevalidated methods and SOPs for automating a growing list of chemistry kits on the Hamilton MICROLAB STAR liquid handling platform.
Validating third-party chemistries
Sorenson Forensics provides forensic DNA laboratory services for the validation of serology, extraction, quantification, and amplification procedures (STR and Y-STR) as well as instrumentation, robotics, training courses, and process flow efficiencies. Sorenson Forensics’ staff is experienced in validating public crime labs as well as private DNA testing labs, and the company’s laboratory director has served on the SWGDAM Subcommittee for Validation. At the completion of a forensic DNA lab validation, the company assists customers with staff training to implement the new procedures and integrate them into current workflow.
Since entering into the strategic partnership with Hamilton, the Sorenson Forensics’ team has validated many of the leading third-party forensic chemistries on the Hamilton MICROLAB STAR platform including all single tandem repeat (STR) and real-time PCR amplification kits for DNA analysis. Additional methodologies such as DNA extraction and normalization/concentration are being verified and validated on a continual basis.
Hamilton automated liquid handling workstations have two distinguishing features that make them particularly well suited to application in the forensic lab. The first is the company’s unique air-displacement pipetting, which delivers measurable advantages over liquid-filled positive displacement systems.
Air displacement pipetting reduces the chance of crossover contamination and eliminates system fluid dilution effects sometimes seen in liquid-filled platforms. Furthermore, air displacement pipetting allows for the incorporation of pressure sensors in the pipetting channels, which serve to monitor each pipetting step, giving users the opportunity to react to problems like empty samples, foam or clots immediately.
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Figure 2: TADM software screenshot showing where common aspiration and dispense errors can be diagnosed along the pressure curve. (Source: Hamilton Robotics) |
Hamilton’s novel Total Aspiration and Dispense Monitoring (TADM) technology monitors pipetting steps in real time, ensuring that probative DNA samples are processed correctly and their integrity maintained. Once the TADM profile of a given liquid is established, curve limits can be set (Figure 1). The MICROLAB STAR can be programmed to automatically handle events when these limits are exceeded. (Figure 2). TADM verifies with a traceable digital audit trail that a sample has been successfully transferred, the documentation of which could be valuable in an analyst’s court testimony. TADM replaces exhaustive pipetting studies that are required to confirm the pipetting performance of other liquid handlers.
Confirming performance When considering the automation of a DNA chemistry kit, the forensic lab needs confirmation that the automated procedure performs equal to or better than manual methods—especially with regard to sensitivity, reproducibility, contamination control, and sample tracking. Sorenson Forensics’ validation protocols meet or exceed SWGDAM guidelines and are designed to thoroughly evaluate the performance of new robotic protocols while being flexible enough to suit the needs each individual laboratory. Studies commonly include experiments to test sensitivity, reproducibility, concordance, inhibitor effects, and contamination.
One of the latest chemistries that Sorenson Forensics has validated on the Hamilton STAR platform is the
Promega PowerPlex 16 HS STR kit, which was recently approved by the FBI for use in laboratories participating in or generating DNA records for the bureau’s National DNA Index System. Sorenson conducted a complete verification and validation study to assess the robot’s ability to use this kit in forensic casework. Liquid handling was shown to be accurate and precise through visual verification and comparison to manually processed samples. Results obtained from repeated samples across different runs showed excellent reproducibility, sensitivity, and reliability while contamination monitoring showed that sample integrity was not compromised throughout the automated process. Hamilton’s TADM feature confirmed proper performance of liquid pipetting and provided a documented record of successful sample transfers for all runs.
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Figure 3: Typical plate layout example used to determine contamination and reproducibility. (Source: Hamilton Robotics) |
Profile data from duplicate amplifications of all 10 NIST 2391 SRM DNA sample extracts matched the published results at all loci tested. The integrity of sample tracking was maintained without error by Hamilton’s integrated bar-coding tracking system. These results indicate that a single source target DNA amplification amount of 1.5ng was sufficient to generate optimal heterozygote peak height ratios (Figure 3). Other studies to test and validate the system included: analysis of mixtures and non-probative casework samples and a manual comparison.
Additionally, Sorenson Forensics observed that the Hamilton liquid handling provided more precise use of reagents and reduced the overage need common in other automated procedures. This resulted in a substantial savings in reagent costs.
With this extensive validation process and evaluation already completed, a forensic DNA laboratory can quickly implement automation of the PowerPlex 16HS kit on the Hamilton STAR with optimized methods from Sorenson. The Hamilton/ Sorenson collaboration eliminates time-consuming validation work on the part of the forensic lab by providing bundled automation systems and validation services.
About the Authors
Rick Luedke, marketing manager, is an employee of Hamilton Robotics.
Darren Warnick, PhD, validation engineer, and Craig Nolde, validation services manager, are employees of Sorenson Forensics.
This article was published in Bioscience Technology magazine: Vol. 34, No. 4, April, 2010, pp. 12-15.