AutoLys workstation: Validation of a fully automated forensic sample lysis solution

Transcription

1 AutoLys workstation: Validation of a fully automated forensic sample lysis solution Application note Top 3 reasons for automation of this application Sample lysis and DNA extraction can be regarded as quality bottlenecks in the DNA profiling process, influencing the amount, quality and traceability of forensic samples. The AutoLys workstation is the first fully automated system offering a real solution to overcome the common risks occurring in forensic laboratories. Fully automate sample lysis Ensure complete traceability throughout the process Optimize lysis efficiency and DNA yields Introduction Working closely with forensic institutes worlwide has led Hamilton to the development of a solution to help solve one of the bottlenecks of the DNA profiling process. The AutoLys system is a combination of a smart tube design and the automation of the forensic sample lysis process. Forensic DNA profiling starts with the DNA extraction of a biological trace (e.g. a blood stain) on a trace carrier material (e.g. a cotton swab). The DNA is extracted from the biological trace. The AutoLys workstation automates this early step of the DNA profiling process. For crime scene samples it is crucial that as much DNA as possible is retrieved from a trace, since quality and quantity are not known beforehand. A suboptimal technique might cause loss of quality to a potentially already low quality crime scene trace. DNA extraction can be regarded as the quality bottleneck for further downstream processing. At the same time, the process of forensic DNA extraction of crime scene samples has a high risk of user errors. A high number of samples per batch and many different sample types with many sample transfers from one tube to another make fail-safe automation highly desirable. The Netherlands Forensic Institute (NFI) is an external agency of the Dutch Ministry of Security and Justice developing the most useful and advanced forensic products and services and supplying them to a wide range of national and international clients. As an ISO/ IEC accredited laboratory it runs a quality management system which is accredited by the Dutch Accreditation Council in a number of fields since The NFI has performed the developmental validation of the AutoLys workstation and AutoLys tubes according to the ISO/IEC standard, using simulated crime scene samples. Particular emphasis was put on DNA extraction efficiency and on error-free processing. Figure 1: Hamilton s AutoLys STARplus in the NFI laboratory.

2 Highly efficient and fail-safe DNA extraction from forensic traces In order to optimize the laboratory workflow and improve traceability as well as DNA yields the sample lysis and DNA purification are performed on the same platform, thus enabling the full automation of forensic DNA extraction from crime scene samples. System description The AutoLys system used for the validation project is a AutoLys STARplus with eight independent pipetting channels and four independent channels for AutoLys tube handling (see Figure 2). It is equipped with an Autoload for automated loading of samples and a high resolution 2D barcode reader. Five AutoLys heater shaker modules, a centrifuge with swingout rotor, carriers for AutoLys tube racks, caps, output tubes, reagent reservoirs and tips are included on the deck. Loading Figure 2: AutoLys channels inserting four AutoLys tubes into an AutoLys tube rack. AutoLys tubes are tube-in-tube consumables for sample lysis and collection of the soluble DNA lysate by centrifugation. The sample is placed in the inner tube which is separated from an outer tube by a filter. The tube is designed for optimal heat transfer in the AutoLys heater shaker and lysis takes place with the inner tube fully inserted into the outer tube. After lysis, the inner tube can be lifted for retrieving the lysate into the outer tube by centrifugation. The AutoLys channels automate all AutoLys tube handling: de-capping, re-capping, lifting and locking the inner tube, removing the inner tube and placing it back. The AutoLys racks in SBS format allow parallel processing in 24 tube (4x6) format. Up to four racks containing 24 tubes each can be loaded on the system. A 2D barcode on each AutoLys tube provides full sample id traceability. Figure 3 shows AutoLys tube assembly and the position of the barcode. Figure 3: AutoLys tube components

3 Method description For lysis the biological material is immersed in a buffer containing enzymes. DNA in the cleared lysate is then purified by binding to a substrate (e.g. silica beads or a silica column) and repetitively washed by using special cleansing buffers. Finally, the purified DNA is eluted from the substrate in a small volume of an elution buffer. A small portion of the DNA extract is used to generate a DNA profile. First, the concentration of the DNA in the extract is determined, followed by amplification of specific regions (so-called DNA markers ) of human DNA in the extract, using a short tandem repeat polymerase chain reaction (STR-PCR). These amplified markers are then analyzed by capillary electrophoreses, resulting in the peak pattern that makes up the DNA profile. The final step in this process is the analysis of the DNA profile, using specialized analysis software. To assess the performance of the AutoLys system, the NFI directly compared results from samples processed using the AutoLys system to results from current manual extraction using the QIAshredder columns (QIAGEN). Subsequent DNA purification from both series of lysates was carried out manually following a NFI validated protocol. DNA extracts were then quantified (Quantifiler Duo DNA Quantification Kit, Life Technologies) and analyzed by STR typing (AmpFlSTR NGM Amplification Kit, Life Technologies). Figure 4 shows the manual DNA extraction procedure. The workflow for the automated process is visualized in Figure 5. Figure 4: The manual DNA extraction procedure. With the large number of samples processed there is a high risk of introducing human error. Validation Figure 5: Workflow for AutoLys tubes processing on the AutoLys workstation. The entire procedure is depicted in the upper segment, detailed lysis steps in the AutoLys tubes are shown in the lower segment. Validation included the following steps: The performance of the AutoLys tube and the AutoLys heater shaker versus a conventional lysis tube and shaker with respect to DNA yield. The performance of the automated DNA lysis process using AutoLys tubes on the AutoLys workstation in comparison to the manual method using conventional spin columns. Cross contamination was checked and DNA yield was evaluated. As a measure for DNA extraction efficiency, quantitative real-time PCR (qpcr) of DNA markers and analysis of peak heights in electropherograms of DNA profiles were used. Quality of extracted DNA was assessed by calculating two parameters: Mean local balance (MLB) values which are based on intralocus peak balance for all markers within a DNA profile and IPC values for the presence of inhibitors (qpcr).

4 Results AutoLys heater shaker efficiency Temperature is critical for the DNA extraction process. An efficient heat transfer from a heated shaker to the sample tube is critical. To illustrate DNA yield of samples processed in AutoLys tubes on the AutoLys heater shaker it was directly compared to that of samples processed in conventional tubes on the conventional shaker. All liquid handling was done manually and qpcr was performed for comparing DNA yield. It was found that the AutoLys heater shaker has a significant positive effect on the yield of the DNA extraction (Figure 6). This effect is particularly beneficial for blood samples (average yield ratio of 233.7%), but also visible for saliva samples (average yield ratio of 117.3%). It demonstrates that the AutoLys system is superior to a conventional shaker in the overall lysis procedure due to a better heat transfer from the AutoLys heater shaker to the AutoLys tubes. Cross-contamination Hamilton and the NFI have developed an optimal automation protocol to prevent cross-contamination during DNA lysis. This protocol guarantees that no sample-switching can occur and dictates specific pathways of the robotic arms and channels, preventing movement over open tubes and reagent troughs. The event of cross-contamination was evaluated through DNA profiling. Three dedicated experiments were performed, using specific plate layouts (see Table 1). AutoLys tubes containing forensic traces were alternated with empty tubes (blanks). A fourth experiment to test for cross-contamination was the continuous analysis of a blank sample prior to and after a sample batch during regular validation runs, therefore testing for Blanks 0% (0/500+) crosscontamination with each run performed on the instrument. This way, more than 50 additional tests were performed. As can be seen in Table 1, there has not been any indication for crosscontamination caused by the AutoLys system. Figure 6: Two series of 7μl blood (undiluted) and 50μl saliva (2x diluted) on cotton swabs were lysed using AutoLys tubes on the AutoLys heater shaker or conventional tubes on a conventional heated shaker. Separation of lysate and trace carrier for all samples was performed in QIAshredder homogenizers (QIAGEN). Cross-contamination test Layout Result Checkerboard 0% (0/40) Zebra-stripes horizontal 0% (0/40) Zebra-stripes vertical 0% (0/40) DNA yield and quality A variety of sample types were tested that are commonly found on crime scenes. The biological traces blood, saliva and epithelial traces were depositeded on typical trace carriers in a high and a low concentration. The AutoLys system worked with all tested and commonly found crime scene samples. As Table 2 shows, lysis on the AutoLys system is highly efficient. The performance of the AutoLys system is expressed as overall yield and given as percentage of the results from the AutoLys system versus results of the manual method, based on peak heights from DNA profiles. Table 2 lists the results for all samples, a graphical summary is given in Figure 7. The yields from the AutoLys system were comparable to or better than those achieved with manual extraction. The quality indicators MLB ratio and IPC ratio as direct comparison of the respective values for the automated and manual process show that the DNA extracts from the AutoLys system were of high quality. The DNA profiles were clean with no inhibitors present. [DNA] in ng/µl Blood Saliva Conventional shaker AutoLys heater shaker Table 1: Results from cross-contamination testing. Results are depicted as percentage of samples in which cross contamination was detected. The overall sample number is shown in parentheses.

5 Material Sample (concentration) Sample preparation Overall yield MLB ratio IPC ratio Cotton swabs (n=10) Terry cloth (n=10) Polyester (n=10) Denim (n=10) Fleece (n=10) Used or consumed samples (n=10) Touch trace samples, tape-lifted (n=20) Blood (low) ~ ng/µl 5µl (75 x diluted) 114.2% n.d. n.d. Saliva (low) ~ ng/µl 15µl (30 x diluted) 125.7% n.d. n.d. Blood (high) ~ ng/µl 5µl (15 x diluted) 96.9% Blood (low) ~ ng/µl 5µl (225 x diluted) 113.9% Saliva (high) ~ ng/µl 15µl (3 x diluted) 89.4% Saliva (low) ~ ng/µl 15µl (90 x diluted) 91.3% Blood (high) ~ ng/µl 5µl (15 x diluted) 71.2% Blood (low) ~ ng/µl 5µl (225 x diluted) 127.1% Saliva (high) ~ ng/µl 15µl (3 x diluted) 121.6% Saliva (low) ~ ng/µl 15µl (90 x diluted) 100.9% Blood (high) ~ ng/µl 5µl (15 x diluted) 101.8% Blood (low) ~ ng/µl 5µl (225 x diluted) 113.4% Saliva (high) ~ ng/µl 15µl (3 x diluted) 111.1% Saliva (low) ~ ng/µl 15µl (90 x diluted) 94.1% Blood (high) ~ ng/µl 5µl (15 x diluted) 112.9% Blood (low) ~ ng/µl 5µl (225 x diluted) 84.7% Saliva (high) ~ ng/µl 15µl (3 x diluted) 108.0% Saliva (low) ~ ng/µl 15µl (90 x diluted) 102.4% Cigarette butts ~ ng/µl n.a % Cigar butts ~ ng/µl n.a. 63.1% Chewing gum ~ ng/µl n.a % Envelopes ~ ng/µl n.a % Stubs ~ ng/µl n.a. 89.2% Table 2: DNA yield from samples lysed on the AutoLys system. Overall yield is given as percentage of manual lysis yield with sample numbers given in the material fields. The first quality indicator MLB ratio should approach 1.00 or be higher indicating that there is no difference between the series. MLB is the intralocus peak balance for series of samples. It is defined as the ratio between the height of the 300% lower peak and the height of the higher peak in a heterozygous marker, giving a marker-specific 250% ratio between 0 and 1. High values mean high quality of DNA extract. The second quality 200% parameter IPC ratio should be 1.00 or lower indicating that DNA extracts processed on the AutoLys system contain comparable or 150% lower levels of inhibitors than DNA extracts Blood (high) processed. manually. 100% Blood (low) 50% 0% Saliva (high) Saliva (low) Normal usage Figure 7: Overview of tested crime scene samples in this validation study. Results are shown in percentage yield of automated vs. manual lysis.

6 Conclusion Hamilton and the NFI have collaborated in successfully validating the AutoLys workstation and AutoLys tubes in a forensic environment. The AutoLys system can efficiently be used to perform the lysis and subsequent separation of a wide variety of forensic traces without loss of yield and/or quality when compared to conventional methods. Overall, an average yield of 112.1% has been determined. This means that in average the AutoLys system could extract 12.1% more DNA from a forensic sample, whilst maintaining the same level of quality of the DNA extract compared to manual extraction reflected by the absence of inhibitors and quality of DNA profiles, but without all risks that are involved with manual DNA extraction. In addition, the AutoLys system was able to perform the lysis of crime scene traces without inducing (cross-)contamination. The results from the various materials in different DNA concentrations demonstrate that the AutoLys system is a powerful and reliable solution for DNA extraction from biological samples in general. System specifications AutoLys STARplus 8+4 workstation, eight pipetting channels, four AutoLys channels, Autoload, centrifuge, 2D barcode reader, five AutoLys heater shakers, carriers for AutoLys tubes, caps, output tubes, reagent reservoirs and tips System dimensions: Width: 2160mm Height: 903mm Depth: 1006mm Labware requirements Provider AutoLys tubes Hamilton Reagent reservoirs Tips Hamilton Hamilton Acknowledgements We would like to thank Bas de Jong and Maureen Antonie, Department of Human Biological Traces, Netherlands Forensic Institute for codeveloping this method and kindly providing the data used in this application note. Figure 1 and Figure 4 are property of the NFI Hamilton Robotics GmbH. All rights reserved. Lit. No. AN-XXXXXX QTY: 600, XX/13 Printed in Germany. Web: USA: infoservice@hamiltonrobotics.com United States Tel: United Kingdom & Ireland Tel: +44 (0) Brazil Tel: +55 (11) China Tel: France Tel: +33 (01) Italy Tel: To find a subsidiary or distributor in your area, please visit hamiltonrobotics.com/contacts. Denmark, Norway, Sweden, Finland Tel: +46 (0) Germany, Switzerland, Austria, Benelux Tel: +49 (0)