Autologous Bone Marrow Transplantation - Blog Science Connections

Immunomagnetic Purging ofBurkitt's Cells 445
marrow cell samples (10 x 10 6
cells/ml) were then incubated with 2-mg
beads/ml for 30 minutes at 4°C under continuous slow rotation and passed
through the magnetic system. The number of beads depends on the total
number of cells to be treated, whether normal or malignant, and 2 mg of beads
are then needed to clean samples containing less than 10% malignant cells.
The second step of the procedure involving the incubation with the beads
and the magnetic separation was repeated twice (cells were then resuspended
in appropriate medium for the freezing procedure).
Except for a few experiments in which malignant cells were detected with
the Hoechst staining method (5), we used a liquid cell culture assay. After
treatment, samples initially contaminated with 1% BL cells were grown in a
liquid culture, and the percentage of growing BL cells at day 10 compared to a
calibration curve, enabling us to quantify the number of residual BL cells after
treatment. If no BL cell is detectable at day 10, samples are kept 2 more weeks in
culture before we conclude that all BL cell growth has been inhibited, a finding
usually correlated to the complete elimination of these cells in this assay (6; see
also the above-named chapter by I. Philip et al. in this volume).
Mean results were then expressed in decimal logarithms of the initial ratio
of malignant and normal cells divided by the final ratio after the purging
procedure. In individual experiments, results are expressed as percentages of
growing BL cells in the culture at day 10.
RESULTS AMD DISCUSSION
As shown in Table 1, using the cocktail of three MAbs—B^ Y29/55, and
J 5
—in this procedure enables us to eliminate 4-5 logs of malignant cells from
samples contaminated with BL cells from five different cell lines, three having
recently been established from our patients' tumor cells. In 11 of 22
experiments, malignant cells are fully eliminated. However, in three experiments,
Table 1. Efficiency of the Modified Procedure
Cell Experiments Depletion Burkitt's Lymphoma Cell
Line (n) (log) Growth Inhibition
Daudi 4 4 2/4
Raji 3 4 2/3
BLg 3
5 5 5/5
F3L 2
5
4a
2/5
(1 incomplete: 3 logs)
BL 99
5 4" None
(2 incomplete: 3 logs)
Note: Sample contamination was 1%. Burkitt's lymphoma cell elimination is
expressed as a mean of three to five experiments on each line. Full elimination has
been controlled at day 21 of culture.
"Mean.