Autologous Bone Marrow Transplantation - Blog Science Connections

352 Burkitt's Lymphoma Purging Assay
MATERIALS AND METHODS
Normal bone marrow obtained from healthy donors or from patients with
Burkitt's lymphoma was collected on preservative-free heparin, diluted in
phosphate-buffered saline (PBS) without calcium and magnesium (Gibco), and
separated on a Ficoll gradient (Lymphoprep [ 1.077 density], Niegaard, Norway).
Four BL cell lines—IARC BL 1 7
, lARC BL 9 3
, IARC BL 2
, and lARC BL 99
—have
recently been established from our patients' tumor cells, and their characteristics
have been previously described (1,4,5). Fresh Burkitt's cells were obtained from
pathological bone marrow, the effusions, or the initial tumors.
The assay was carried out under sterile conditions. In the artificial model,
allogeneic normal mononuclear bone marrow cells were irradiated (50 Gy) and
contaminated with BL cells from various cell lines. In the autologous model,
patients' mononuclear bone marrow cells were contaminated with BL cells
from their own tumors. The BL cells were added in five tenfold serial dilutions to
samples of 4 x 10 6
BM cells: the initial and final BL cell concentrations in
triplicate samples being 10 2 (4 x 10 4 total BL cells) and 10 6 (4 total BL cells).
The last triplicate cultures contained a single BL cell. The triplicate samples
from each dilution were poured into 5 ml of RPM1 medium supplemented with
20% heat-inactivated irradiated fetal bovine serum, glutamine, and antibiotics in
Corning 25100 plastic culture flasks (vertical position with 10 cm 2 MRC 5
feeder
layer of embryonic irradiated fibroblasts) (Merieux, Lyons, France) and
incubated at 37° C in 5% C0 2
in air. The cultures were checked twice weekly—
we observed them with an inverted microscope and changed half of the
medium. On the tenth day, samples were taken and the percentage of growing
BL cells was evaluated in each aliquot by cytology on a cytocentrifuged smear.
When no BL cell was seen on the smears, the culture was kept for 2 more weeks
and checked again each week until the demise of the cells. Selected cultures
with growing BL cells were also propagated for 3 weeks in order to show that the
features of growing BL cells (Epstein-Barr virus-associated nuclear antigen
reactivity, immunologic markers and cytogenetics) remained identical to that of
the initial cell line used for the contamination (1-3).
Requirements for removing BL cells from excess bone marrow with two
immunologic methods, the rabbit complement lysis procedure and the
immunomagnetic purging procedure, have been extensively described elsewhere
(6-8; see also Combaret et at "Eliminating Burkitt's Cells From Excess
Bone Marrow With an Immunomagnetic Purging Procedure," in this volume).
RESULTS
Four representative lines recently established from our patients' tumors
were chosen from a panel of 100 BL cell lines for their distinct characteristics,
including lines that were EBV negative or positive that were from North African
or Caucasian patients. Lower BL contaminations were studied with three to five