When subculture volumes exceed 3 liters, the fresh medium used is AIMV. 
Cultures growing in serum-containing medium are thus diluted into AIMV, and 
no further LAK supernatant is added to cultures growing in serum-containing 
or serum-free medium. 
If, during the growth of TIL, patients performance status has 
deteriorated to 3 or greater or if they have developed significant cardiac, 
renal, pulmonary or hematologic dysfunction than they will be taken off study 
and will not receive the infusion of TIL or IL-2. 
When the total lymphocytes for a patient are ready for harvest, 5 x 10 6 
cells are taken for cytological examination. Cytospins are examined for the 
presence of remaining tumor. At least 200 cells are studied and therapy 
proceeds only when no tumor cells are found. Other lymphocyte samples are 
taken for characterization of cell surface markers and for assessment of 
cytotoxicity using techniques identical to that in our previous protocol 
(86-C-183; reference 23, attached). Briefly, lymphocytes are stained with 
fluorescent-labeled antibodies (Leu2, Leu3, Leu4, Leu7, Leull, Leu5, Leu9, 
LeuM3, HLADDR and Tac). Chromium release assays are performed with K562, 
Daudi autologous tumor, and allogeneic tumor targets. 
To infuse the lymphocytes they will be thawed and grown for one to three 
additional weeks using the same procedures detailed above. For infusion 
TIL are reharvested. At the time of cell collection, one liter of saline 
for injection is pumped through the collection chamber and the centrifuge 
is stopped. Lymphocytes are resuspended in the collection bag, the centrifuge 
is started again, and another liter of saline is pumped through to fully wash 
the free of tissue culture medium components. The cells are then filtered 
through a platelet administration set into 600 ml transfer packs (Fenwal), and 
50 ml of 25% albumin and 450,000 IU of IL-2 are added to the 200 to 300 ml 
volume of cells in saline. The TIL are infused over 30 to 60 minutes through 
a central venous catheter. 
7. Interleukin-2 : The recombinant IL-2 used in this trial will be 
provided by the Division of Cancer Treatment, National Cancer Institute 
Recombinant DNA Research, Volume 15 
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