Core D: Work Flow

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Core D Work Flow
Core D Prepare kits for Blood collection
Core D Blood processing
Sample shipment to Coriell
Functional indicators Project 3
FTA cards for Project 1 genetics
Clinical Chemistry (Lab Corp)
Nutrition Parameters (In-house other)
Core E data repository
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Blood Collections from Centenarians

• 5 ml lavender tubes (Project 1)
• 2 10 ml yellow tubes (Coriell)
• 5 ml lavender tube
• Reference sample
• Plasma nutrition parameters
• Clot Project 1
• 3 ml lavender tube
• Lab Corp clinical chemistry
• 7 ml red top tube serum
• Clinical chemistry
• Nutrition

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Project 1 genetics Whole blood spotted onto FTA
cards
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8-10 FTA cards spotted per subject
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FTA cards (cont.)

• Dried samples taped shut, packaged in individual
  envelopes
• Samples packaged in 6 X 9 envelopes
• 4-5 cards / envelope
• 2 envelopes per subject
• Samples shipped to Michal Jazwinski in two
  shipments

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Clinical Chemistry

• 3 ml whole blood and 2 ml serum shipped by
  courier to Lab Corp
• Measure
• Complete Blood Count (19 parameters)
• Comprehensive Metabolic Prolife (17 parameters)
• T4 and TSH
• Hemoglobin A1c
• C-reactive protein
• Ferritin

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Clinical Chemistry (cont.)

• Receive results by Fax (next day)
• Fax results to Dr. Lisa Tenover (project
  physician)
• Contact Sandra for patient/family info for
  abnormal results
• Enter data in Excel files and transmit to Core E

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Nutrition Parameters

• Individual serum plasma samples stored at 80
• 715 Boyd Grad (Hausman lab)
• 380 Dawson Hall (Johnson lab)
• Assays performed when sufficient samples
  collected (25-40)
• Results entered in Excel files and transmitted to
  Core D

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Coriell Institute for Medical Research
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Fedex at the door
Blood samples arrive at Coriell in the morning.
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Samples are checked-in
Samples are logged in by a Data Manager. Paper
work is carefully checked.
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Repository Information Management System
This system keeps track of all data about all
samples. The computer will assign a Reference
Number to each sample and track the sample
throughout Coriell.
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Bar-coding the Samples
A computerized bar code is generated which
contains information about the sample. One bar
code label is placed on each sample tube and one
is placed on the paperwork.
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Bar code label is attached to the paperwork
This makes it easy to follow the sample and it
prevents errors.
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Blood Sample goes to the Lab

• We want to make a cell line from a type of white
  blood cell .
• To separate red blood cells from white blood
  cells the blood is diluted and placed over a
  special solution in a tube.
• This tube is spun in a centrifuge. The red cells
  go to the bottom and the white cells go to the
  middle.
• This is shown in the next few slides.

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Diluted Blood is Layered over Separating Solution
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Cells are Separated in a Centrifuge
Cells are spun for 30 minutes at a gentle speed.
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Lymphocytes form a Tight Band
The red arrow points to a band of
lymphocytes. The red blood cells are in the
bottom of the tube.
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The Lymphocyte Band is Carefully Removed
The lymphocyte band is removed to a tube, the
cells are washed, and resuspended in culture
medium.
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Making a Cell Line from Lymphocytes

• Lymphocytes will die in culture after a
  relatively short time. They dont divide
  forever.
• By infecting the cells with Epstein Barr Virus,
  the culture can become immortal. This will
  provide cells and DNA for scientists for as long
  as they are needed.

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Immortalizing the Lymphocytes
The virus is added to the culture together with
an agent to encourage growth. These cells will
be cultured until there are enough cells to
freeze.
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Cell Culturing
Cells are grown in an incubator at body
temperature (37oC). As they grow they are
transferred to bigger flasks.
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When we need lots of cells we grow them in roller
bottles. One roller bottle is equivalent to 80
little flasks. These will be used for making DNA.
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Cells Cultured in Roller Bottles
Cells in roller bottles are slowly rotated to
ensure good growth.
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Preparation and Shipment of DNA

• Most Human Variation samples are shipped as DNA.
• DNA is stored in freezers at -70oC it is stable
  for many years.

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Technician prepares DNA
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DNA Samples are Prepared for Freezing
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DNA Samples are Stored in Racks in -70oC Freezers
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Each Freezer can Hold 32,000 Ampules of DNA
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Frozen Storage of Cell Lines

• Cell lines can be frozen and thawed for later
  use. Freezing in liquid nitrogen (-170oC) will
  preserve cells so they can be revived after 30-40
  years!
• Freezing must be done very carefully.
• This process is shown in the next few slides.

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Cells are Frozen in Glass Ampules
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Ampules are Flame Sealed
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Ampules are Stored on Canes
After sealing the ampules they are placed on a
cane and sent for testing and freezing.
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Ampules are Tested to See if They Leak
Before freezing the ampules are tested to see if
they leak using a blue dye. A leaking ampule can
explode or contaminate the cells.
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Freezing Cells
Cells are frozen in a special freezer that
reduces the temperature slowly and steadily.
Frozen cells are then transferred to a liquid
nitrogen tank.
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The Tank Room
Liquid nitrogen is kept in insulated tanks. The
tanks are subdivided into compartments.
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A Liquid Nitrogen Tank
The ampules of cells are kept completely
submersed in liquid nitrogen. Tanks are
monitored frequently to ensure normal levels of
liquid nitrogen. Backups are kept at two other
locations.
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A Liquid Nitrogen Tank
Each cane, with 5 ampules, is put in a canister.
The location is recorded in the Repository
Database.
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Coriell Ships Cells Worldwide