selection or after the CD19 negative selection. The 
relative effectiveness of the CD34 versus the CD19 
selection will be evaluated at the time of relapse by 
examination of the leukemic cells in the patient at the 
time of relapse. If the patient has the LNL6 but not the 
GINa, it will be clear that the CD34 purge is not 
complete enough. Whereas the CD34 followed by the CD19 
negative selection will be sufficient for the removal of 
the leukemic cells from the marrow, the CD34 selection 
alone is not sufficient. 
Thus, the retroviral marking can be used to assess the 
relative contribution of the two purging techniques for 
removal of neoplastic CLL cells. 
INFORMATION TO BE COLLECTED FROM THE ANALYSIS: The 
double PCR strategy outlined in this protocol for 
analysis of the outcome from the technical point of view 
permits unambiguous interpretation of the experimental 
results. The splitting of the cDNA products into two 
halves and the double amplification strategy, one half of 
the sample used in a sequence amplification reaction for 
actin and the immunoglobulin gene rearrangement, and the 
other half amplified with primers for the neo gene and 
the immunoglobulin gene rearrangement, will allow us to 
identify four types of cells following transplant and at 
the time of relapse: 
1. The normal cell is not transduced by virus. The only 
positive amplification product will be the actin. 
The interpretation is that the purging procedures 
were successful, the transduction was flawed 
technically, or there was clonal evolution so as to 
favor neoplastic cells from the marrow or systemic 
circulation which were not transduced. 
2. The normal cell is transduced by virus. This cell 
shows that the transduction worked at least for the 
normal cells. The culture of the cells exposed to 
the transduction protocols in assay systems which are 
specific for the late, intermediate, and early 
progenitors, coupled with the retroviral marking, in 
vitro fractionation, and bone marrow transplantation, 
will provide information about the efficiency of 
procedures designed to isolate the early progenitor 
cells and about the value of the fractionation 
procedures for the isolation removal of leukemia 
cells and isolation of the early progenitor cells. 
3. The CLL cell is not virally transduced so that the 
amplification reaction for the actin and for the 
immunoglobulin rearrangement are positive, but both 
of the virus reactions are negative. This indicates 
that the purging or the systemic therapy was 
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