Autologous Bone Marrow Transplantation - Blog Science Connections

ABMT in Acute Nonlymphocytic Leukemia 61
methods (12). For each patient, an attempt was made to obtain at least 4 x 10 8
nucleated marrow cells per kilogram of body weight.
Approximately 70% of the collected marrow was treated ex vivo with 4-HC
at a concentration of 60 /^g/ml in one patient in second remission, 80 /ig/ml in
one patient in second and in one patient in third remission, and 100 jug/ml in
the remaining 37 patients. The mean number (± 1 SD) of nucleated marrow
cells thus treated was 3.0 + 0.47 x 10 8 /kg (range, 1.6-5.3 x 10 8 ). In 33 patients,
the remainder of the collected marrow was treated with a lower dose of 4-HC (40
or 60 M9./ m l) or remained untreated as a reserve marrow to be infused in the
event that engraftment did not occur with the fully treated autologous marrow.
Because of suboptimal collection, seven patients had no reserve marrow
available.
The buffy coat fraction was removed from the collected marrow suspension
by centrifugation in standard blood transfer packs in a Sorvall RC 3B centrifuge
with an HG-4L head at 2,900 rpm for 10 minutes; the process was then repeated
to obtain a second buffy coat layer. These two fractions of nucleated cells were
pooled and mixed with autologous plasma and heparinized tissue culture
medium (TC 199) (GIBCO, Grand Island, NY) to obtain a concentration of 2
x
10 7 cells/ml. A solution of 4-HC was freshly prepared in phosphate-buffered
saline, and an appropriate volume was added to the marrow cell suspension to
obtain the desired final concentration of the drug. The cells were incubated with
4-HC in a water bath at 37°C for 30 minutes, after which the cell suspension was
rapidly cooled to 4°C and centrifuged for 10 minutes. The 4-HC-treated cells
then were resuspended in 45% TC 199, 45% autologous plasma, and 10%
dimethyl sulfoxide at a concentration of 4
x
10 7
cells/ml. Aliquots (50 ml) of the
cell suspension were placed in polyolefin bags, frozen in a controlled-rate
freezer at -l°C/minute to -50°C and at -10°C/minute to -70°C, and
transferred to the liquid phase of a liquid nitrogen freezer. At the time of marrow
infusion (designated as day 0), each bag was rapidly thawed in a 37°C water
bath, and the thawed cell suspension was infused through a central venous
catheter at a rate of 10-15 ml/minute.
Hematopoietic Progenitor Cell Assays
For each patient, 4-HC-treated marrow samples were assayed for granulocyte-macrophage
colony-forming units (CFCIs-GM) in a soft agar culture
system, as previously described (10). In brief, marrow mononuclear cells were
cultured in 35-mm plastic tissue-culture dishes that contained 0.3% agar, 15%
fetal bovine serum, and McCoy's 5A medium; human placenta-conditioned
medium was added as colony-stimulating factor. After incubation for 10-14
days at 37°C in 7.5% C0 2
in humidified air, culture dishes were examined under
magnification (X35-40) for the presence of granulocyte-macrophage colonies.
Aggregates of more than 40 cells were considered colonies. The total number
of CFCls-GM infused into each patient was calculated from the dose of
nucleated marrow cells infused and from the frequency of colonies in culture.