3) TNF production by the producer line will be measured and should be 
significantly above baseline control values. TNF will be assayed using 
standard biologic assays on the L929 sensitive cell line (Asher et al. J. Immunol 
138:963-974, 1987) on or by ELIZA assay (R&D Systems, Minneapolis, MN). 
4) Sterility of the producer line and the supernatant will be assured 
by testing for aerobic and anaerobic bacteria, fungus and for mycoplasma. 
5) Viral testing will be performed including: 
a. MAP test 
b. LCM virus 
c. Thymic agent 
d. S+/L- assay for ecotroplc virus 
e. S+/L- for xenotropic virus. 
f. S+/L- for amphotropic virus. 
g. 3T3 amplification. 
6) Electron microscopy will be performed to assure the absence of ad- 
ventitious agents. 
The retroviral supernatant will not be used to transduce TIL infused 
into patients until approval is received from the Food and Drug Administration. 
6. Tests on the transduced TIL population . Following transduction and 
growth of the TIL populations the following tests will be performed on the TIL 
prior to Infusion into patients. 
1) Cell viability will be greater than 70% as tested by trypan blue dye 
exclusion. 
2) As in all prior TIL protocols, cytologic analysis will be performed 
on over 200 cells prior to infusion to assure that tumor cells are absent. 
3) Sterility will be assured by testing for aerobic and anaerobic 
bacteria, fungus and mycoplasma. 
4) S+/L- assay including 3T3 amplification must be negative. 
[274] 
Recombinant DNA Research, Volume 14 
