In-house Method and Process Development
Novel CTC Markers using CTCs on Filters (Sarcoma Pilot Study)
The Sarcoma pilot study aims to enumerate CTCs from the blood of patients who have been diagnosed with Osteosarcoma, Ewings Sarcoma or Rhabdomyosarcoma. ISET (Isolation by Size of Epithelial Tumour Cells) is a filtration system that enriches CTCs whole from blood based on the assumption that CTCs are larger than leucocytes. The leucocytes pass through the pores leaving larger cells and CTCs on the surface of the filter.
A four colour immunofluorescent assay has been developed to identify CTCs in a background of leucocytes from patients diagnosed with Osteosarcoma. A Fluorescent In Situ Hybridisation (FISH) assay is being developed using break-apart probes that identify specific gene translocations for Ewings Sarcoma and Rhabdomyosarcoma.
Imaging Capability within CEP
The ImageStream X mk II captures images of objects in flow, a powerful technique able to combine the speed, sensitivity, and phenotyping abilities of Flow Cytometry with detailed imagery and functional insights of microscopy. It produces multiple high resolution images of every cell directly in flow, including brightfield, dark field (SSC) and up to 10 fluorescent markers. Applications include the enumeration of rare cells or objects, the characterization of circulating tumour cells, cell cycle analysis, analysis of intracellular co-localisation and translocation, as well as many others.
Ariol automated microscope: allows 4 colour fluorescent image capture and FISH relocation on slides. Works at up to 60x magnification with the addition of brightfield capability, and Ariol associated analysis software.
Bioview automated microscope: capable of imaging IHC slides in brightfield with associated binning software. Capable of imaging slides in 4 colours and re-location of desirable cells, at up to 60x magnification. It is also suitable for subsequent fluorescent institute hybridisation (FISH) in rare cells. Bioview associated software and Definiens compatible.
Via the Advanced imaging and flow Cytometry group CEP also has access to:
PerkinElmer Opera Phenix, a spinning disk based confocal screening system with a dedicated sCMOS camera per detection channel (4). Magnification from 1.25-x60 (water and air). Automation allows loading of up to 42 multi-well plates at room temperature or from an incubator. Columbus software compatible.
PerkinElmer Vectra: automated multi-spectral fluorescent imaging system with compensation facility, capable of scanning slides up to 7 fluorescent labels. Image output is Definiens compatible.
Aperio Versa: capable of automated imaging of slides at 5x to 60x magnification and precision scanning of brightfield and fluorescent (4 colours), with the accuracy and resolution required for FISH. Columbus compatible. Software is in place for 3D histology reconstruction.
Leica gSTED super resolution microscope: the ability to image at molecular levels of resolution. The system utilises multiple lasers to image to a resolution of 25nm. Software tools are available for image processing (Huygens) and Image Analysis (Imaris).
Multiple software programs are employed including: Definiens, IDEAS, Imaris, FlowJo and Columbus.
Additional resources available via the Advanced Imaging and Flow Cytometry group include: Multiple Flow Cytometry sorters and analysers the majority of which can be carried out at Class II containment, Widefield, confocal and two-photon microscopy, and Organoid imaging.
The Laboratory Information Management System (LIMS)
CEP has seen its clinical biomarker portfolio rapidly expand with biomarkers deployed on several clinical studies. During this time the inventory of GCP accredited scientific equipment and validated biomarker assays has also exponentially increased. In order to maintain and improve our regulatory compliance levels, CEP successfully secured funding to cover the purchase of a Laboratory Information Management System (LIMS). LIMS software offers key features that support and streamline a modern laboratory's operations, and provides us with the infrastructure required to support the complexity of modern trial design and downstream sample processing.
Working closely together with the system vendor LabWare (http://www.labware.com) we have configured and validated our LIMS to support CEP’s many and varied trial related activities. The final system encompasses project management, sample tracking, work planning, instrument interfacing, data transfer and acquisition, auditing and reporting. All samples are tracked within LIMS from their arrival within CEP until they are disposed, providing us with a complete chain of custody. Tests are scheduled and results stored directly with LIMS and where possible direct data transfer links have been established with instruments to improve data integrity. We currently have three analysis platforms configured for direct data upload; Dynex absorbance plate reader, Aushon multiplex ELISA platform and the Triturus automated ELISA platform. All other results can either be manually entered or transferred from spread sheets into LIMS via copy/ paste functions or a generic data upload facility. A key aspect of the configuration work has been to build upon the quality systems already in place within CEP, by providing a structured environment to plan work and store data. The additional functionality provided by LIMS has enabled us to ensure that all data routinely exported has undergone a strict two-step review process to ensure the best quality data is provided to the clinical trials and experimental medicine studies we support. The final system has been released in phases to ensure the functionality is made available as quickly as possible while satisfying the computer system validation requirements. A bespoke, modular training package has been established to ensure users are trained only in the work streams required by their roles. The training is conducted in a dedicated training system to protect the main trials database, and has so far been delivered to in excess of 50 users.
The improvements in efficiency and throughput provided by LIMS have increased our capacity to conduct clinical biomarker studies within CEP. LIMS development will focus on emerging technology platforms and keeping pace with assays under development and validation, ensuring that LIMS remains relevant to our areas of research focus. The internal expertise we have gained since 2009 will ensure that the system fully meets operational needs now, and in the future, to support the collaborative nature of CEP. The LIMS facility will enhance the world class treatment and research facilities within MCRC already supported within CEP.