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Category
I. Cytomics
II. Genomics
III. Pharmacokinetics
IV. Xenografts
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Services
AMPL is a fee for service facility based on a cost recovery business model to provide competitive pricing for customized one-stop-shopping for the molecular profiling of specimens.
Users are required to complete a Work Request Form W1.
All projects are reviewed by a Steering Committee with a focus to support methodological developments and to study disease pathology and therapy response.
AMPL staff are qualified and trained in applicable standard operating procedures (SOPs) for the collection, storage and processing of biospecimens, the application of materials and methods, and the calibration and maintenance of equipment. AMPL is guided by NCI Best Practices for Biospecimen Resources and good laboratory practice (GLP) procedures to assure quality and consistency of the biospecimens and data quality.
Specimen Handling
The use of biospecimens must adhere to applicable ethical requirements; additional review by the Research Ethics Board of the University Health Network may be required.
AMPL is equipped to process wet tissue, frozen tissue, fixed/paraffin-embedded tissue and blood. The simultaneous histo-pathological analysis of multiple specimens is performed with the construction of tissue microarrays (TMAs). Regions of interest for molecular analyses are extracted using laser assisted micro-dissection and microscopy.
I. Cytomics
Histology, Immunolabeling, and Image Capture & Analysis
Immuno-labelling techniques for immunohistochemistry and immunofluorescence analyses are optimized for specific marker and tissue type.
Fast multi-spectral imaging for whole tissue sections is imaged using a three laser TISSUEscope™ confocal scanning system (Biomedical Photometrics Inc.). For cellular detail, higher resolution images are acquired using conventional fluorescence microscope.
Immunohistochemistry-stained sections are imaged using a whole slide scanner (ScanScope CS, Aperio Technologies Inc.) that belongs to AOMF, a collaborative imaging core facility at the Princess Margaret Hospital.
Advanced image analysis applies tailor-made work processes supported by in-house image analysis software and commercial software including Image-Pro Plus (Media Cybernetics Inc.) and MCID (Interfocus Ltd.).
Flow Cytometry
Real-time pharmacodynamics analyses of single cell signal activation use advanced clinical flow cytometry applications. Experiments are optimized on either a Cytomics FC 500 (Beckman Coulter, Inc.), or a LSRII (BD Biosciences) supported by a collaborative core research flow cytometry facility at PMH.
ELISA
Protein concentrations in biospecimen fluids are quantified by enzyme-linked immuno-sorbent assays (ELISA) using commercially available kits and customized in-house kits.
II. Genomics
FISH
Gene copy is determined using the fluorescence in situ hybridization (FISH) technique with use of gene specific-labeled probes available commercially (Vysis Inc.), or by custom-labeled bacterial artificial chromosome (BAC) clones. FISH image analysis is done using an Axioplan 2 imaging system (Zeiss). A Metasystem workstation which includes an automated Zeiss microscope is also available to provide a diversity of software modules for classical cytogenetic analysis, manual & automated metaphase and interphase FISH, and high-throughput manual & automated tissue microarray acquisition & analysis.
Gene Mutation Detection (DNA/SNPs/RNA)
Several methods are employed to maximize the nucleic acids yield and quality from the specimen type received. A fully automated nucleic acid extractor (Qiagen BioRobot M48) provides a fast and efficient approach to isolate nucleic acid from specimen, handling up to 48 samples at once. Isolated nucleic acid can subsequently be used in PCR based analysis. A Genetic Analyzer 3100 (Applied Biosystems) provides a platform for high throughput sequencing analysis for mutation detection. Mutation discovery is also performed using nucleic fragment analysis using a dHPLC system (Transgenomics Ltd.). Quantitative real-time PCR (ABI 7900HT, Applied Biosystems) allows the detection of mutant transcription level among cells and SNPs.
III. Pharmacokinetics
Drug and metabolite analyses in biospecimens are performed using the latest chromatography instruments that include HPLC-UV and LC-MS/MS mass spectrometer. The LC-MS/MS system is a triple quad (MDS Sciex API 32000, Applied Biosystems) with Electrospray, TurboIonSpray & APCI ionization probes. The LC-MS/MS system provides high sensitivity for quantifying small molecules in pg/ml to ng/ml concentration ranges. Data analysis is performed with WinNonLin.
IV. Primary Tumour Xenograft Models
AMPL has expertise in the establishment of primary xenografts grown orthotopically / subcutaneously in severe immunodeficient mice, and their use to test therapeutic agents.
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