Sample Preparation

DOE-SPEC-1142-2001

6.1.2. The tubes should be labeled with patient's name, date and time blood is

drawn, identification number, and name of person obtaining specimen at a

minimum.

6.1.3. Do not refrigerate the blood specimen or the packing materials. Shipping

containers should be labeled: HUMAN BLOOD DELIVER

IMMEDIATELY. DO NOT FREEZE, PERISHABLE. Biosafety packing

should be utilized.

6.2.

SAMPLE PREPARATION

6.2.1. Upon receiving a sample, separate the lymphocytes by centrifugation on a

density gradient medium using a method that will maximize the yield of

mononuclear cells. Aspirate the cell interface with a sterile pipette. Wash the

cells three times in approximately 10 ml of buffered saline. Perform a cell

count to determine the amount of complete medium to add so that the final

cell concentration is 2.5 X 105 cells/0.1 ml. Each well will receive 0.1 ml of

the cell suspension and 0.1 ml of mitogen, antigen, beryllium concentration or

complete medium to bring the well volume to 0.2 ml per well.

6.2.2. The recommended plate arrangement requires approximately 6ml of cell

suspension (concentration of 2.5 x 106/ml) distributed into 4-12 wells each of:

6.2.2.1.

at least 4 treated wells for each of the 3 concentrations of BeSO4,

to be harvested on two separate days, from day 4 to day 7;

6.2.2.2.

8 to 12 Control wells, 0.1 ml/well (cells in complete medium only,

harvest correspond to harvest days chosen in 6.2.2.1). If two sera

are used, then control wells will be seeded using each serum;

6.2.2.3.

4 Mitogen/antigen #1wells (harvest on the optimal day); and

6.2.2.4.

4 Mitogen/antigen #2 wells (harvest on optimal the day).

6.2.3. Incubation for the number of days selected shall be carried out at 37C in an

atmosphere of 5-10% carbon dioxide-air.

6.2.4. At 6 to 18 hours prior to each harvest, add 1 Ci tritiated thymidine to each

well. Return plate to incubator.

6.2.5. Harvest cultures using a 96 well harvester and filter mats. Cells will be

collected in a standard glass filter fitted harvester or its equivalent and counts

measured in a beta emission scintillation (using a suitable cocktail) or gas

ionization counter expressing the data in cpm. Counting time should be at

least 1 minute per tube if a beta emission scintillation counter is used and 4

minutes per filter if a gas ionization counter is used.