7.0 BONE MARROW HARVEST, CELL SEPARATION AND CRY OPRES ERVATI ON 
7.1 Peripheral Blood Stem Cell Collection 
Peripheral blood will be processed through a Fenwall 
CS3000 blood cell separator via continuous flow 
centrifugation, and non-mononuclear blood components 
returned to the patient.. Collections will begin when 
the total white blood cell count reaches 1000/ul after 
cyclophosphamide 4 gm/m 2 and G-CSF mobilization (see 
Section 6.2) and will continue daily until a total 
nucleated cell count of at least 3 X 10 8 cells/kg is 
reached. Aliquots of < 5 X 10 7 harvested cells will be 
taken from each days collection to assess progenitor 
content, surface antigen expression, and for in vitro 
studies of tumor involvement and retroviral 
infectivity. The remaining cells will be frozen in 10% 
DMSO , 20% autologous or ABO homologous serum and media 
in a controlled rate freezer and stored in liquid 
nitrogen. 
7.2 Bone Marrow Harvest 
Patients will have marrow harvested using aseptic 
technique from the posterior and anterior iliac crests 
under general anesthesia in the operating room. 
Multiple marrow aspirations will be collected in 
heparinized syringes and held in a Fenwall bone marrow 
collection unit. Volumes totalling 1000-2500 cc will be 
collected, with a yield of at least 0.5 X 10 8 nucleated 
cells per kilogram body weight pre-processing necessary 
to go onto high-dose melphalan and total body 
irradiation autologous transplantation. 
7.3 Bone Marrow Processing 
Marrow will be filtered through the Fenwall collection 
kit ^f inters to remove bopy particles and transported to 
the blood bank for further processing in sealed, 
sterile collection bags. The total bone marrow will be 
processed with a Fenwall CS3000 automated separation 
unit using the albumin. method to give a mononuclear 
cell fraction. Samples totaling < 5 X 10 7 cells will be 
used for counting, in vitro culture, microbial culture, 
analysis for tumor involvement, retroviral infectivity 
and surface antigen analysis. 
7.4 Cryopreservation of Marrow and Peripheral Blood Cells 
After processing of the cells as above, a solution 
.containing 10% , DMSO plus, autologous plasma in McCoy's 
Recombinant DNA Research, Volume 16 
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