Pre-IDE Submission: Clinical Protocol 
Neuroblastoma Bone Marrow Purging System 
BAXTER HEALTHCARE CORPORATION, HYLAND DIVISION 
Immunomagnetic Purging Procedure 
All procedures will be performed using aseptic technique. One half of the marked 
mononuclear marrow cell preparation corresponding to a single vector will be 
resuspended in 20-50 ml (minimum safe volume) of sterile normal saline for infusion 
containing 10% v/v autologous plasma or albumin. To this suspension is added 1 mg 
each of anti-neuroblastoma monoclonal antibodies UJ13A, 5.1H11, M3 40, 127.11 and 
THY1. The cells are incubated with the antibodies for 30 minutes at 4°C, with mixing 
by gentle swirling every ten minutes. Following incubation, the volume of the cell 
suspension is increased to approximately 200ml by addition of cold (4°C) buffer. The 
cells are pelleted by centrifugation at 250g for 8 minutes at 4°C. The supernatant buffer 
is aspirated, and the washing procedure repeated until the cells have been washed a total 
of four times. The final cell pellet is resuspended by gentle swirling in 50-55ml of cold 
(4°C) buffer. 
During the incubation of marrow cells with the antibodies, the Dynabeads® M-450 Sheep 
Anti-Mouse IgG ST (Baxter Code 4R5451) are prepared. The number of beads required 
is equal to the total number of nucleated cells being processed and is calculated from the 
following formula: 
Vol. of beads to be added in ml 3 Number of nucleated cells 
Bead Concentration (beads/ mL)* 
This volume is aseptically removed from the vial and transferred to a 50ml polypropylene 
tube. Fifteen milliliters of buffer is then added to the bead suspension, and the beads 
collected by placing the tube into the MPC-1 magnetic separator (Baxter Code 4R4513) 
for 3 minutes. The supernatant buffer is aspirated and discarded, the tube removed from 
the separator, an<i the washing procedure repeated twice more. The final bead pellet is 
resuspended in 15 ml of buffer. 
* The Dynabeads™ are supplied by the manufacturer in a solution which contains 0.02% sodium azide as a 
preservative. Prior to use in toxicology studies, this solution is discarded and the beads are rinsed three times 
in the appropriate vehicle. The amount of residual azide after the third wash is calculated to be £250 pg/mL. 
According to RTECS, there is no known bioreactivity of sodium azide at the picogram level. (Study No. 92002- 
PT-011) 
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Recombinant DNA Research, Volume 16 
