De Blasio

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					           AINP 42nd Congress
       May 24 - 27th, 2006 Rome Italy

      Corso Teorico Pratico
    Tissue and Brain Banking
The European Huntington Disease Network

       Speaker:      Ing. Pasquale De Blasio
     “Coriell - BioRep” Partnership
Global Biorepository “service providers”

      Pasquale De Blasio, CEO
      BioRep S.r.l.           Via Fantoli 16/15 20138 Milan Italy           e.mail:
                     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 services
to 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 Institute
for 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.
                    Coriell-BioRep Locations

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

• 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
     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
Coriell –BioRep information System

             Coriell - BioRep
            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
            Central Vs. Local BioBanking

                       Virtual Network
    - Local Storage            - Central Network

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

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

   High Q supports HD Clinical Research through the
                   Huntington Project

      Cohort                                 Registry
 Huntington Project                     Euro HD Network
                  USA                                   Europe

                  Central Biorepositories
Coriell Institute for            BioRep               Medical
         Research, NJ-USA                 Milan, Italy

   USA - Canada                      14 European Countries
90 Centres involved                   104 Centres involved
  Collection sites                      Collection sites
          Current collection kit
• Contains all items required to package and send
• Packaging and shipping cost per subject approx 40
  euros ($33) (exc. labour and overheads)
                     • Comes flat-pack reducing shipping costs
                     • Relatively cheap & easy to use

                     • 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.
       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)
                      • 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

                      • High shipping costs
                      • Bulky
                      • Packaging procedure more complex
EHDN Registry Repository

        May 2006
• 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
  – 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.
• 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
  – 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
• 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.

• 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.
                    EHDN – SAMPLES RECEIVED

              Registry repository contains 69 subject samples

                         EHDN REGISTRY REPOSITORY


              25                                               Madrid

              20                                               Lisbon
              15                                               Barcelona
              10                                               Ulm
               5                                               Graz
                   Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06

Approx 30 additional centres in next 3 months

 Region / Countries   No. Centres          Locations
    Scandanavia           4         Oslo, Bergen, Turkku, Lund
       Benelux            1                  Lieden
      Denmark             1               Copenhagen
         Italy           1+4              Milan + 4 sites
   Spain & Portugal       2             Valencia, Granada
         UK               6             Cardiff & 5 others
      Germany             10             To be confirmed
     Switzerland          1                    Bern

• 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
BioRep Cell Culture Laboratorie
             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
• Remote Fail-Safe Storage
   – cell lines
   – DNA
   – blood products and other biomaterials
BioRep Molecular Biology Laboratories
BioRep Molecular Biology Laboratories
               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
                        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
          Coriell - BioRep Cell and Tissues 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
                                 •   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
                                      – 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
                   Vendita Prodotti e Controllo
      BioRep Sistema di MonitoraggioBioRep

                                  • Ethernet, RS232, e Wirless
                                  • 24/7 - 365 days/years
                                    continuous monitoring and
                                  • Alarm system based on 3 levels
                                    of alarms (visual, sound, SMS)
• Monitoring and Control System   • Web visualization
                                    accessible with Password
                                  • Certification reports (charts,
                                    tables, etc.)
            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
Biorepository Management System
                     BioRep web portal

• Access through Biorep web- portal (
• User self registration
• Subscribed User
  – Purchase services & biomaterial from catalog
                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)
                              The Tissue MicroArrayer


   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

                          Galileo TMA CK3500   -   Integrated Systems Engineering srl, Milan, Italy
The Tissue MicroArray (TMA) technology

                       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
                                      TMA Design

 Software assisted

 Performed remotely by the arrayer
                       TMA Design

                            To optimise the tissue core placement
                                    for subsequent analysis

                                                          Normal Tissue
                       Lesion                             (near lesion)

                                                          Case X

                                                          Case Y

                                                    Normal Tissue
                          Normal Tissue
                                                  (resection margin)
                          Control Tissue
TMA construction
The sampling procedure
                                  TMA analysis
                       SEL1L anti-body expression evaluation

Case Number      L    L   L     L    L   NM NM NM NM NM NM NM NM NM
               SN 1 SN 2 SN 3 SN 4 SN 5 SN 1 SN 2 SN 3 SN 4 SN 5 SNv1 SNv2 SNv3 SNv4
    1            0    0   1     0         0    0   ND ND     0
    2           ND ND     0    ND ND      0   ND    0   0    0
                                                              SEL1L expression intensity
    3            0    0   0    ND         0   ND    3
    4            2   ND   3     0    0    1    2    1   1    0 0      Negative
    5            2    1   1     1    1    2    2    1   1      1,2,32 Positive
    6            0    0   0     0    0    2    2    2   1                 1     2
                                                                3+ 1 Plasma cells (Reference)
    7            2    2   2                                         1     1    ND
    8            2    2   0     3
    9            1    1   1    ND                                   0     2
    10           2   ND   2     2   ND                             ND     0     0    ND
    11           0    0   0     0    1    0    0    0   0           0     0     1     0



            Not sampled

         ND Not Defined

                     Milano, 16 giugno 2006
Sala Conferenze
BioRep srl
Via Fantoli 16/15
Milan (MI) - Italy
Thank you for your collaboration and kindness
            from the BioRep team

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