Optimizing Donor Matching With Automated HLA Typing

| 1 CASE STUDY COMPANY CAMPAIGN Summary Anthony Nolan is a leading institution in the Histocompatibility and Immunogenetics (H&I) Field. The H&I Laboratories at Anthony Nolan provide Human Leukocyte Antigen (HLA) typing services for Stem Cell transplantations, as well as disease association studies, pharmacogenetics, and the development of personalised therapies. In this Case Study, we focus on describing the recently automated NGS-based method for HLA typing. This United Kingdom Accreditation Service (UKAS)-certified method leverages the flexibility of GenDx’s NGSGo kits and Hamiton’s automated liquid handling systems and the power of Illumina’s sequencing and analysis technologies to provide high-resolution HLA typing of 1-11 loci in up to 20,000 samples per year. About Anthony Nolan Anthony Nolan is a UK-based charity that works in the field of blood cancers and disorders and stem cell transplantation. The charity is named after Anthony Nolan, who did not suffer from leukemia but from Wiskott–Aldrich syndrome, a rare genetic blood disorder. Doctors advised that the only form of treatment was a bone marrow transplant, but at that time, no register of potential donors was available. Anthony’s mother, Shirley, established the Anthony Nolan Bone Marrow register in 1974. Sadly, no match for Anthony could be found. He died at the age of seven in 1979. Since then, Anthony Nolan’s registry and associated services have grown and made a significant impact worldwide, with over 22,000 patients receiving lifesaving transplants and more than 800,000 people registered as potential donors. Lisa Walsh, Director of Laboratory Operations at Anthony Nolan, explained the charity mission: “We aim to connect patients in need with generous volunteers who are willing to donate their stem cells. For individuals with blood cancer or a blood disorder, a stem cell transplant from a donor could be their best chance of survival. Anthony Nolan is dedicated to finding the best possible match for each patient.” With over 350 employees working across three sites (The Anthony Nolan Research Institute in London, The Anthony Nolan Cell Therapy Center in Nottingham, and the London Histocompatibility and Immunogenetics (H&I) Laboratories), Anthony Nolan supports patients both in the UK and abroad. Lisa Walsh highlights: “We are committed to transforming the lives of those needing stem cell transplants through research, embracing new cell therapies, and providing HLA typing services. As a founding member of Bone Marrow Donors Worldwide (BMDW) and the World Marrow Donor Association (WMDA), Anthony Nolan has facilitated connections between patients and donors worldwide.” Anthony Nolan is also renowned for maintaining the world’s only central database of HLA alleles, solidifying its leading position within the H&I community. Learn more about Anthony Nolan Authors: Adam King1 , Franco Tavarozzi1 , Selda Duman1 , Lisa Walsh1 , Dimitri Michalakis1 , Adam Elliston2 , Carolina Elejalde3 , Gabriela Boza-Moran3 * 1 Anthony Nolan; 77B Fleet Rd, London, NW3 2QU, United Kingdom 2 Hamilton Sales & Services UK LTD, Unit 1 Forge Mills Park, Station Road, Birmingham B 46 1JH, United Kingdom 3 Hamilton Bonaduz AG (Robotics), Via Crusch 8, 7402 Bonaduz, Switzerland * For correspondence: mboza-moran@hamilton.ch Automating NGS-Based HLA Typing for Stem Cell Transplantations CASE STUDY ANTHONY NOLAN NGS IN HLA TYPING Figure 1: Timeline of Sequencing-Based HLA Typing Technologies used at the H&I Laboratories at Anthony Nolan. | 2 HLA Typing at Anthony Nolan The H&I Laboratories offer services to donors and patients awaiting Haematopoietic Stem Cell (HSC) transplants, as well as to research organisations and healthcare providers. Franco Tavarozzi, Head of HLA Typing and Development at Anthony Nolan, further explained the role of the H&I Laboratories: “In order for a transplant to take place, a donor who closely matches the patient must be found. Matching is based on the Human Leukocyte Antigen (HLA) type, which is identified by sequencing the patient’s HLA genes alongside those of potential donors.” The HLA Typing services offered by Anthony Nolan are also used for various applications, such as disease association studies, pharmacogenetics, and the development of personalised therapies. Accredited by the United Kingdom Accreditation Service (UKAS) to ISO15189:2012 and the European Federation for Immunogenetics (EFI), the H&I Laboratories have a team of over 50 staff members and utilise automated laboratory equipment to process and sequence DNA samples on a daily basis. Automation of Molecular HLA Typing Methods The Anthony Nolan H&I Laboratories have been performing HLA typing through various methods for over 30 years, keeping up to date with emerging technologies and methods. Franco Tavarozzi described the evolution of the technologies and instruments used in the H&I Laboratories at Anthony Nolan over the past decade: “In 2013, we purchased the first two Hamilton Microlab® (ML) STARs™ to facilitate a semi-automated Sequence-Based Typing (SBT) workflow. Two years later, we decided to move to Third-Generation Sequencing (TGS) with Pacific Biosciences’ Single-Molecule Real-Time (SMRT) sequencing technology and acquired two new Hamilton ML STARs™. In 2020, we moved to Next-Generation Sequencing with Illumina’s Sequencing by Synthesis technology while leveraging GenDx NGSGo kit’s flexibility to select the HLA loci we wanted to amplify and sequence for each sample. We have now automated the GenDx-IIlumina method with our Hamilton liquid handlers and can currently run automated NGS-based HLA typing” (Figure 1). 2013 2X Hamilton Microlab® STAR 2015 2X Hamilton Microlab® STAR 2013 Sanger Sequencing 2015 Single-Molecule Real-Time (SMRT) Sequencing 2020 Sequencing by Synthesis Sequence-Based Typing (SBT) Thermo Fisher Scientific ABI Genetic Analyzer Third-Generation Sequencing (TGS)-Based Typing Next-Generation Sequencing (NGS)-Based Typing PacBio RSII Illumina MiSeq / iSeq The Services provided by Anthony Nolan include: • Stem Cell Donor Registry HLA typing. • Clinical Service HLA Typing, including support for solid organ transplantation. • High Throughput DNA extraction. • Virology Testing for HIV, HepB, HepC and CMV. • ABO RhD blood group typing. • HLA Antibody screening and identification. • Genetic mutation tests (e.g., HFE for Haemochromatosis risk) and CCR5 (HIV resistance). • Support for researchers and developers requiring extra laboratory resources or specialist expertise. Learn more about Anthony Nolan’s services. Figure 2. Pre-PCR Hamilton Microlab® STAR™. (1) Tips stackers; (2) Filtered Tips; (3) 3X Cooling Carriers with Adaptors for 96-Well Plates, Deep Well Plates and Microtubes; (4) easyCode Carrier; (5) CO-RE® Gripper; (5) Barcode Scanner; (7) 1ml CO-RE® Channels; (8) 96-Multi-Probe Head. 1 2 3 4 5 6 7 8 | 3 Automated Library Preparation for NGS-Based HLA Typing Adam King, Automation Laboratory Scientist at the H&I Laboratories at Anthony Nolan, explained the reasoning behind the automation of the GenDx-Illumina workflow: “The automating of our workflow allows us to cover three main needs: 1.) Reduction of manual intervention and associated errors; 2.) Processing of larger runs of up to 95 samples (from the current maximum of 32 samples per run) and 3.) Higher method flexibility by significantly facilitating the amplification and sequencing of different HLA loci in each sample.” To avoid potential amplicon contamination, the complete workflow is performed in two distinct areas; Pre-PCR and Post-PCR. System Overview Anthony Nolan has four Hamilton ML STARs™, two of which are already set up to run the GenDx-Illumina workflow and two of which are currently not in use and kept as backups. One of the active systems is in the Pre-PCR area, and the other is in the Post-PCR area. Both systems have eight 1000μL pipetting channels and a CO-RE® 96 Multi-Probe Head and use CORE® II conductive tips to leverage Hamilton’s conductive Liquid Level Detection (cLLD) across all methods. The deck configuration, however, differs. In the Pre-PCR system (Figure 2), three Hamilton Cooling Carrier Modules provide controlled-temperature positions at 5 °C for plates and tubes. The system also has an easyCode Carrier for reading 2D-barcoded DNA sample tubes and an Orbit Barcode Scanner for labware control. The Post-PCR system has two Cooling Carriers, one Hamilton Heater Shaker (HHS) for incubations and shaking steps, and a magnetic plate for bead-based clean-ups (Figure 3). Description of the Automated Workflow The NGS-Based HLA Typing workflow uses GenDx NGSgo®- AmpX v2 kit for high-resolution HLA typing of 11 loci: HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1, including all exons for HLA DRB1 and whole gene primers for HLA DQB1. The complete workflow is divided into ten steps. (Figure 4): 1. Sample reception 2. DNA extraction 3. Loading and checking of DNA samples’ identity 4. PCR set-up 5. Amplification of 1-11 HLA loci 6. Amplicon verification and quantification 7. HLA loci pooling 8. Library preparation 9. Sequencing 10. Data analysis Adam King described the workflow in more detail: “After the samples are received, their DNA is extracted using Promega’s Maxwell® RSC Nucleic Acid Purification Kits, Maxprep™ Liquid Handler (based on Hamilton’s ML NIMBUS) and Maxwell® RSC instruments. The DNA samples are subsequently loaded into the Pre-PCR ML STAR, and their identity is checked by the 2D-Barcode Reader. Then, Pre-PCR plates are prepared in the automated system using the primers of the GenDx NGSgo AmpX v2 kit for the amplification of 1-11 HLA loci. Amplification is performed in an Applied Biosystems™ Veriti 96-well Thermal Cycler. The amplified fragments are then verified and quantified using gel electrophoresis and a Qubit Fluorometer in the Post-PCR lab. Next, the HLA loci are pooled together, and the GenDx NGSgo Library Preparation is carried out in the Post-PCR ML STAR. The prepared libraries are then subjected to sequencing, using either Illumina MiSeq or iSeq. Finally, the data generated from the sequencing is analysed, using Illumina Sequence Analysis Viewer and GenDx NGSengine software.” Workflow Implementation and Validation To develop, implement, and verify the automated method, Anthony Nolan worked in close collaboration with Hamilton Application Specialists. Two variants of the method were developed: one for low-throughput, based on the use of the 1ml channels and one for high-throughput, leveraging the use of the 96 Multi-Probe Head. Adam Elliston, Application Specialist at Hamilton, explained the process: “After each method was programmed and all syntax was correct, they were tested in simulation to ensure that the logic of the automation was accurate, including plate movements, pipetting steps, and any necessary calculations. Water testing was then conducted to check for general pipetting accuracy and that appropriate liquid level detection and error handling were in place. Tests with real liquids were then used to ensure transfer volumes were true and adjust liquid classes, as required. Following this, biological testing was carried out to check that the outputs at each stage of the process were comparable to the existing manual processes.” Several tests were carried out to validate the method. The most critical ones are described in Table 1, along with the criteria for acceptance. - Test 1 focused on validating the low-throughput method, meticulously testing 16 samples across 11 loci. The results were carefully compared to manually-processed samples to ensure the accuracy of the automated approach. - Test 2 centered on validating the high-throughput method, examining 95 samples for two specific loci. Similar to the low-throughput method, the results were compared to manually-processed samples. - Test 3 addressed the need for business continuity, ensuring that the methods could be executed seamlessly without input from a worklist. This demonstrated that the methods could be run, even in the presence of network issues through custom dialog boxes in the VENUS software. - Test 4 ensured that the output file generated after a run would contain all the necessary information for sample traceability and further processing. 1 2 3 4 5 6 7 Figure 3. Post-PCR Hamilton Microlab® STAR™. (1) Tips; (2) 3X Cooling Carriers with Adaptors for 96-Well Plates, Deep Well Plates and Microtubes (3) Multiflex Carrier with Reagent Trough Carrier, Tips and 1X Hamilton Heater Shakers; (4) Tip Stackers; (5) Barcode Scanner; (6) CO-RE® Gripper; (7) 1ml CO-RE® Channels; (8) 96-Multi-Probe Head. 8 Test Acceptance Criteria Test 1 – 16 Sample Run – 11 Loci 95% Pass rate at amplification Library quantification between 10 – 100nM All SAV metrics within defined ranges 100% Concordance with previous HLA typing Test 2 – 95 Sample Run – 2 Loci Test 3 – Run without Dispatch File The user must be able to run all methods without an input file, with no errors. Test 4 – Run in simulation mode The user must be able to run all methods in simulation mode to generate the same output file as a live run. Table 1: Validation of the Automated NGS GenDx-Illumina Workflow. | 4 Technical Service Team Pre-PCR Lab Post-PCR Lab 10. Data Analysis 10. Illumina Sequence Analysis Viewer & GenDx NGS Engine 1. Sample Reception 1. Received Samples 2. DNA Extraction 2. Promega MaxPrep Liquid Handler 4. 96-Well Plates for PCR Reaction Amplification of 1-11 HLA Loci 5. 5. Applied Biosystems Veriti 96- well Thermal Cycler 3. DNA Samples in 2-D Barcoded Plate 7. Deck Layout for Pooling 9. Sequencing 9. Illumina MiSeq / iSeq Amplicon Verification and Quantification 6. 6. Gel Electrophoresis 8. Deck Layout for Library Preparation Post-PCR Microlab® STAR Pre-PCR Microlab® STAR 7. HLA Loci Pooling 8. Library Preparation 4. PCR Set-Up Loading and Checking DNA Samples’ Identity 3. | 5 | 6 Adam King commented: “Overall, the implementation and validation of the automated methods took nine months, from developing a business case, scoping the workflow requirements, developing the methods and testing their effectiveness extensively. Using the ML STARs that we had already, only minor tweaks to the deck layouts were required.” Recently, Anthony Nolan obtained an extension to scope from the United Kingdom Accreditation Service (UKAS) to cover the validated automated pipeline. Franco Tavarozzi commented on the relevance of this accreditation; “The extension will allow us to provide Hamilton-automated NGS-based HLA Typing, in accordance with ISO15189 standards. This achievement represents the culmination of our efforts to improve efficiency and accuracy in the laboratory through automation. The lowand high-throughput workflows allow maximum flexibility with changing sample numbers.” Benefits of the Automated Methods Flexibility of the Throughput The H&I Laboratories at Anthony Nolan currently carry out HLA typing on around 5,000 samples yearly, with around ten tests per sample. The automated high-throughput method will allow them to increase this throughput and facilitate its use in research projects and clinical studies with a large number of samples. Furthermore, the low-throughput method gives Anthony Nolan the flexibility to do automated NGS-based HLA typing when the sample number is lower, as an alternative to manual processing. Adam King commented: “Thanks to automation, we now have the capability to process up to 20,000 samples per year, with the option to increase this number if needed. Our Business Development Team is actively pursuing new commercial projects and custom HLA typing requests.” Adaptability of the Automated Systems As Anthony Nolan moved from SBT to TGS and then to NGS, the same Hamilton-automated systems were adapted to meet the requirements of each new workflow. Adam Elliston commented: “Hamilton Liquid handlers are very versatile. Although the implementation of new methods demands skills and time, very often, the only thing that is required from a hardware point of view is the changing of position of the carriers on-deck. Moreover, we can always add new modules if needed, and the ML STAR can be upgraded to the ML STARPlus for a larger deck space.” Flexibility of the Methods When automating laboratory workflows, the goal of achieving standardisation and ease of use often needs to be reconciled with the need to be adaptable and open to adjustments of current methods and the implementation of new ones. Adam King commented on Hamilton Systems’ capability to do precisely that: “My favourite thing about Hamilton Systems is their ability to customise a workflow to match our requirements fully. While plug-and-play laboratory automation with preloaded methods is becoming more popular and has its benefits, laboratories dealing with complex multi-stage workflows find it indispensable to customise their own methods to address unique challenges.” Anthony Nolan is looking to further exploit the flexibility of the automated system by leveraging its cherry-picking capability. Adam King commented: “We are currently working on integrating the automated systems into our Laboratory Information Management System (LIMS). The integration will allow us 1.) To easily perform repeats on specific samples and 2.) To facilitate the selection of different combinations of HLA loci for amplification and sequencing, depending on the sample requirements.” “Reliability, accuracy, and precision are crucial to us. We needed a solution that could easily adapt to our changing work environment, and we have found that with Hamilton Liquid Handlers. Furthermore, having collaborated with Hamilton for many years, we are satisfied with their customer relationship approach and the support they offer. We now look forward to using the automated method in our clinical projects and other service offerings while further increasing the flexibility through the LIMS integration,” concluded Franco Tavarozzi. ©2024 Hamilton Bonaduz AG. All rights reserved. Disclaimer: throughout this Case Study, protected product names may be used without being specifically marked as such. Web: hamiltoncompany.com/automated-liquid-handling Hamilton Bonaduz AG Europe HQ Tel: +41 58 610 10 10 Hamilton Company United States HQ Tel: +1 775 858 3000 United Kingdom & Ireland Tel: +44 121 272 92 80 Spain, Portugal Tel: +34 930 186 262 France Tel: +33 184 008 420 Italy Tel: +39 039 930 0606 Sweden, Norway, Denmark & Iceland Tel: +46 8410 273 73 Benelux Tel: + 31 40 209 178 0 Germany, Switzerland, Austria Tel: +49 89 248 804 804 China Tel: +86 21 6164 6567