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Corso Teorico Pratico Tissue and Brain Banking

AINP 42nd Congress May 24 - 27th, 2006 Rome Italy. Corso Teorico Pratico Tissue and Brain Banking. The European Huntington Disease Network (EHDN) Speaker: Ing. Pasquale De Blasio. “Coriell - BioRep” Partnership Global Biorepository “service providers”. Pasquale De Blasio, CEO

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Corso Teorico Pratico Tissue and Brain Banking

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  1. AINP 42nd CongressMay 24 - 27th, 2006 Rome Italy Corso Teorico PraticoTissue and Brain Banking The European Huntington Disease Network (EHDN) Speaker: Ing. Pasquale De Blasio

  2. “Coriell - BioRep” Partnership Global Biorepository “service providers” Pasquale De Blasio, CEO BioRep S.r.l. Via Fantoli 16/15 20138 Milan Italy www.biorep.it e.mail: p.deblasio@biorep.it

  3. The Coriell Institute for Medical Research For over 50 years the global “Gold Standard” for the storage, procurement, processing, and distribution of both public and private biomaterials for scientific research and discovery. The founder Dr. Lewis Coriell designed breakthrough cell line storage and processing technologies that enabled his colleagues, Drs. Salk and Sabin, to develop the polio vaccine. • For over 30 years Coriell has been the primary provider of cell line repository servicesto the National Institutes of Health (NIH), the NCI and 50 countries around the world. • A sampling of Coriell’s public cell repositories includes collections from National Cancer Institute, the American Diabetes Association and the National Institute on Aging, Juvenile Diabetes Foundations International, National Eye Institute, National Institutefor Allergy and Infectious diseases, National Institute for Arthritis and Metabolic Diseases, National Institute for General Medical Sciences. • With fully certified ISO 9001:2000 cell culture laboratories, Coriell’s ”Best of Class" QA/QC processes, forms, policies and procedures have established the highest scientific standards for cell line, tissue, whole blood and DNA purity, processing and storage.

  4. BioRep is located inside of a New Science Park located in Milan (Italy) near Linate Airport and the major fast carriers sites (DHL, TNT, Federal Express, etc.). BioRepBiorepository Services University of Milan, Centre of excellence CISI Technological Platforms: combinatorial chemistry; HTS genomics; Proteomics; Microarray technology; Bioinformatics and Molecular modeling for human, animal and plant research. MultiMedica MultiLab Multimedica Hospital “core lab” with anatomy pathology and clinical chemistry facilities Integrated Systems Engineering Bio-medical instrument design (Tissue Arrayer, western bloth, etc.) BioSkills organization of specialized accredited ECM (Continuous Education in Medicine) courses for the Biotech industry and Public institutions. Coriell-BioRep Locations • Coriell is located in Camden N.J. USA, next to Cooper Health System Hospital in the Philadelphia try state area. • Mission The purpose of the Coriell Institute for Medical Research is to discover the causes of genetic and non-genetic disorders in order to improve the health of human beings through the banking of cells and tissues.

  5. Coriell-BioRep services • Coriell Institute has a history of achievement and excellence in cell culture technology spanning more than four decades. • Not only has Coriell consistently been in the forefront of developing the fundamental technology of cell culture, it has led the design of electronic catalogues for its Repositories and the implementation of relational databases as key parts of its bioinformatics mission. • Since its founding in 1953, Coriell Institute’s scientists have pioneered in problem’s solutions in cell culture, cell characterization and cryopreservation, thus contributing substantially to the current standard of practice in the field.

  6. Services • Coriell and BioRep offer a range of services to public and private research institutions and are committed to a flexible and innovative approach in order to meet individuals particular needs: • cell culture • molecular biology • cytogenetics • research kits and reagents • cell repository • tissue bank • tissue microarray

  7. Coriell - BioRep Quality Assurance programs • Quality Management • ISO9001:2000 Certification (in progress) • Facility Services GLP/GMP compliant • Testing and Pre-qualifying Reagents • Redundancy of Processing • Redundancy of Storage • Storage in the main facilities (Milan) • Back-up facilities (Monza or USA at Coriell) • Redundancy in the Database • Cell Line and Tissues Quality Control • DNA/RNA Quality Control

  8. Coriell - BioRep Coriell –BioRep information System

  9. BioRep Management System • Biorep Catalog & Order System • Biorep ERP • DHL tracking system • Coriell’s Queue • Third party system already interconnected: • EHDN system, Germany • Coriell, USA

  10. Central Vs. Local BioBanking • Virtual Network - Local Storage - Central Network • Advantages • Direct control of specimens • Immediate availability locally • Small Bio-Bank facility • Easy management of local research group • Disadvantages • Heterogeneity of: • SOP’s • IT infrastructure • Quality of the Bio-Banking facility and organization • Difficult to share specimens • Difficult to manage multi-centres projects • Difficult to expand

  11. Central Vs. Local BioBanking • Virtual Network - Central Storage - Central Network • Advantages • Central Coordination of the projects • Same: • SOP’s • IT infrastructure • Logistics • Large and organised Bio-Banking facility • High quality and Safety standards • Room for expansion • Controlled access to the bio-specimens stored • Disadvantages • The Network needs a formal organization • Long-Term Founding of the Central Bio-Banking facility • Access roles to the bio-specimens

  12. Central Vs. Local BioBanking High Q supports HD Clinical Research through the Huntington Project Cohort Huntington Project USA Registry Euro HD Network Europe Central Biorepositories Coriell Institute for BioRep Medical Research, NJ-USA Milan, Italy 14 European Countries 104 Centres involved Collection sites USA - Canada 90 Centres involved Collection sites

  13. Advantages: Comes flat-pack reducing shipping costs Relatively cheap & easy to use Disadvantages: Limited protection for samples No temperature control - when exposed to 37°C external heat, internal temp raised by 10°C (22 to 32°C) in approx 1hr. Current collection kit • Contains all items required to package and send samples • Packaging and shipping cost per subject approx 40 euros ($33) (exc. labour and overheads)

  14. Advantages: Maintains temp on cold side for 30hrs with external temp of 20°C and 12hrs with external temp of 37°C Greater protection for samples Disadvantages: High shipping costs Bulky Packaging procedure more complex Dual compartment box • One compartment at ambient temperature with 2 gel packs, and one at approx 4°C using 2 frozen gel packs • Packaging and shipping cost per subject approx 94 euros ($78) (exc. labour and overheads)

  15. EHDN Registry Repository May 2006

  16. REGISTRY REPOSITORY • Preparation and disposition of DNA from whole blood for CAG repeat measurement • Isolation of DNA from the whole blood contained in one ACD tube using a modification of the Miller salting out procedure [Miller, S.A., Dykes, D.D., and Polesky, H.F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res. 16: 1215 (1988)]. • Prepare one 25 microgram aliquot of the DNA and dividing the remaining DNA between two additional aliquots (for a total of three aliquots per subject). • Store the 25 microgram DNA aliquot • Measure CAG repeat number.

  17. REGISTRY REPOSITORY • Isolation of lymphocytes and establishment of lymphoblastoid cell lines • Isolate lymphocytes from the whole blood contained in one ACD tube. • Divide the lymphocytes into four aliquots. • Use the aliquots of lymphocytes as necessary to establish a lymphoblastoid cell line from the lymphocytes. • Check the cell line for viability, identity, and freedom from contamination by bacteria, fungi, mycoplasma, and other organism and cell lines. • Prepare 10-20 aliquots of the cell lines. • Store lymphoblastoid cells lines in liquid nitrogen • Store the remaining aliquots of lymphocytes

  18. REGISTRY REPOSITORY • Preparation of DNA from lymphoblastoid cell lines • Expand each lymphoblastoid cell line to produce approximately 200 micrograms of high quality, high molecular weight (>50 kb) DNA. • DNA will be isolated from the lymphoblastoid cells using a modification of the Miller salting out procedure [Miller, S.A., Dykes, D.D., and Polesky, H.F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res. 16: 1215 (1988)]. • Perform quality control studies to estimate of quality and integrity and verify identify. • Prepare 25 microgram aliquots of the DNA • Store DNA as set forth below.

  19. REGISTRY REPOSITORY • Preparation of plasma • Prepare plasma from the whole blood contained in one EDTA tube. • Prepare 0.5 ml aliquots (20 max) of the plasma. • Store plasma as set forth below. • Preparation of urine • For subjects participating in Registry, prepare up to six 4 ml aliquots of urine. • Store urine as set forth below.

  20. EHDN – SAMPLES RECEIVED Registry repository contains 69 subject samples

  21. FUTURE EHDN CENTRES Approx 30 additional centres in next 3 months

  22. DHL DELIVERY TIMES • Av. time from collection to delivery = 25hrs • Longest delivery time = 171hrs (Xmas) • Shortest delivery time = 15hrs • 81% shipments received within 24hrs • 96% shipments received within 48hrs

  23. BioRep Cell Culture Laboratorie

  24. Coriell-BioRep Cell Culture Services • Establishment of cell lines • EBV transformed lymphoblast cultures from human blood • fibroblasts, endothelial, smooth muscle and epithelial cell cultures from appropriate tissue biopsies • Cell banking of public, private, or specialty collections • cryopreservation of primary or transformed cell lines • cryopreservation of peripheral blood mononuclear cells (buffy coats) and selected tissue biopsy material • maintenance of seed and distribution stocks • expansion of cell lines for molecular and cytogenetic analysis • Mycoplasma testing • detection by PCR or microbiological assays • identification of mycoplasma species by immunological or PCR methods • Remote Fail-Safe Storage • cell lines • DNA • blood products and other biomaterials

  25. BioRep Molecular Biology Laboratories

  26. BioRep Molecular Biology Laboratories

  27. BioRep Molecular Biology Services • DNA Isolation • microgram to milligram quantities of high molecular weight DNA isolated from whole blood, cell lines, and tissue biopsies suitable for molecular biology applications • RNA Isolation • polyadenylated (mRNA) and total RNA from a variety of cell lines and tissues verified for quality by RT-PCR • Genetic Mapping • mapping the chromosome location with genetic probes • Whole Genome Amplification (WGA) • high fidelity amplification of DNA from limited amounts of fresh or frozen genomic materials (e.g. from mouthwash samples) • Gender Analysis • gender analysis based on the fluorescent detection of sex chromosome specific alleles • Verification of family pedigrees • genotyping with highly polymorphic microsatellite markers • Promoter Methilation analysis • level of methilation in CpG island • SNPs analysis via DHPL method

  28. Coriell Cytogenetics Services • Coriell's cytogenetic research group has three decades of experience in chromosome analysis and, using the most advanced techniques, is fully equipped for bright field and fluorescence microscopy. Digital imaging and analysis are performed using the Applied Imaging Cytovision Ultra and the Vysis Smart Capture Imaging System. • Karyotyping (Human) • standard analysis of metaphase chromosome using G-banding technique • high resolution analysis of prometaphase chromosomes using G-banding and other banding procedures as required • Karyotyping (Mammalian) • standard analysis of metaphase chromosomes from a wide variety of mammalian species, including but not rrestricted to mouse, dog, pig, as well as mouse stem cells • standard metaphase chromosome analysis of various primate species • FISH Analysis • mapping • localization of chromosome break points • estimation of gene copy number in interphase • determination of interphase zygosity of transgenic mice • Comparative Genomic Hybridization Analysis (CGH) • assessment of chromosome loss, gain, or amplification in a sample of genomic DNA by competitive hybridization of two genomes against a target cell • Chromosome Aberration Analysis and Micronucleus Assays • evaluation of genomic damage by a variety of assays to provide genotoxic end points

  29. Coriell - BioRep Cell and Tissues repositories • The CORIELL and BioRep REPOSITORIES provide essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing cell cultures and DNA derived from cell cultures and tissues. These collections are extensively utilized by research scientists around the world. • Bar-coding and tracking system for all bio-specimen vials • Storage in: • Liquid nitrogen at -196°C or vapour phase (lower than -150°C) • Mechanical (standard and automated) freezers at -80°C (or - 150°C) • Additional storage at +4°C and room temperature • Storage for customer-owned freezers and their contents • PC/PLC based “proprietary” state-of the-art alarms and monitoring system with continuous 24x7 operations. • 100% back-up redundancy / off-site sample storage of all cell and tissues • Remote storage

  30. Vendita Prodotti BioRep BioRep Sistema di Monitoraggio e Controllo Characteristics: • Ethernet, RS232, e Wirless connections • 24/7 - 365 days/years continuous monitoring and recording • Alarm system based on 3 levels of alarms (visual, sound, SMS) • Web visualization (www.biorep.it/servizio) accessible with Password • Certification reports (charts, tables, etc.) • Monitoring and Control System

  31. BioRep Management System • Biorep Catalog & Order System • Biorep ERP • DHL tracking system • Coriell’s Queue • Third party system already interconnected: • EHDN system, Germany • Coriell, USA

  32. Biorepository Management System

  33. BioRep web portal • Access through Biorep web- portal (www.biorep.it) • User self registration • Subscribed User • Purchase services & biomaterial from catalog

  34. BioRep Tissue Microarray Services • preparation of custom tissue arrays and distribution of tissue array slides from specific needs • The new computer controlled tissue arrayer is developed in collaboration with ISE and: • NIH Tissue Array Research laboratory (Dr. Stephen M. Hewith) • Erasmus University of Rotterdam NL (Dr. Peter Riegman) • CNIO of Madrid (Dr. Manuel Morente) • National Cancer Institute of Milan (Dr. Paolo Carbone) • Multimedica MultiLab (Dr. Massimo Barberis)

  35. The Tissue MicroArrayer Specifications • High resolution optics • Automated and computer-guided needles positioning • Automated punching depth regulation • Up-to-6 block automated tray • Dedicated software to assist the user in all operating phases, from the TMA template definition to the final reporting Galileo TMA CK3500 - Integrated Systems Engineering srl, Milan, Italy

  36. Simultaneous histological analysis of hundreds of samples using the minimum amount of reagents. • Use of relatively small areas of tissue (down to 0.6 mm in diameter) • Cost effective technique • Saving of precious biological specimens The Tissue MicroArray (TMA) technology

  37. TMA Design • Software assisted • Performed remotely by the arrayer

  38. TMA_Adenoma Normal Tissue (near lesion) Lesion Case X Lesion Normal Tissue Control Tissue TMA_Adenocarcinoma1 Case Y TMA_Adenocarcinoma2 Normal Tissue (resection margin) TMA Design To optimise the tissue core placement for subsequent analysis

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