Autologous Bone Marrow Transplantation - Blog Science Connections

Magnetic Separation of Marrow Cells 137
School, Durham, ISC), and PM-81 and AML-2-23 were prepared by one of us.
SBA and avidin were purified as previously described (10,11).
Fresh samples of peripheral blood mononuclear cells were obtained by
leukapheresis of patients with acute leukemia. The blast cells were isolated on
Ficoll-Hypaque and incubated with saturating concentrations of MAbs diluted in
PBS (2% FBS) for 30 minutes on ice. After two washes in PBS (256 FBS), the
cells were incubated with 100 jug/ml of FITC and IgG fraction of goat antimouse
immunoglobulin (F1TC-GAM) (Cappel, Malvern, PA) for 30 minutes on ice.
After two washes in PBS (2% FBS) the cells were analyzed for fluorescence
intensity of cells gated on the blast region on either an Ortho 2150 or a Coulter
Epics C. The negative region was set on cells stained with FITC-GAM alone.
Cells were also stained with 100 ug/m\ FITC-SBA.
RESULTS
The data obtained from phenotyping acute leukemia cells is shown in
Tables 1 and 2. Thirty-one cases of acute myelogenous leukemia (AML) and
eight cases of acute lymphoblastic leukemia (ALL) were examined with a panel
of MAbs. The SBA was reactive with most of the AML (11/14) and ALL (4/5)
cases tested, staining a median of 51% of the AML and 70% of the ALL blast
cells. CF-1 reacted with all 31 cases of AML and all 7 cases of ALL, with medians
of 70% and 88% of the blasts staining, respectively. My7 reacted with 23 of 25
AML samples (median, 65%) and PM-81 bound 20 of 22 AML samples tested
(median, 68%). Du-ALL-1 reacted with 8 of 8 ALL cases tested (median, 48%),
J5 bound 5 of 7 (median, 84%) and B4 reacted with 6 of 6 cases (median, 86%).
Reagents were combined in attempts to increase the reactivity of blast
cells. In Tables 3 and 4 the percentage of reactive blasts increased with
combinations of reagents. In one case of M 5
AML, the combination of CF-1,
PM-81, and My7 reacted with 99.6% of the blasts. In one non-T ALL sample, the
combination of CF-1 and J5 reacted with 99% of the blasts. From this analysis,
we chose a cocktail for AML of SBA, CF-1, and MAbs from CD 13 and CD 15,
and for ALL we chose a cocktail of SBA, CF-1, and MAbs from CD 9, CD 10, CD
19, and CD 24. We will use these antibodies for marrow purging.
We examined the efficiency of eliminating specific cell populations using
clonogenic assays. When normal bone marrow mononuclear cells were treated
with a cocktail (OKT-C) of OKT-3, OKT-4, OKT-8, and OKT-11, and MAC, we
examined the elimination of clonogenic T cells using a limiting dilution assay
(Table 5). We were able to remove an average of 2.6 logs of clonogenic T
lymphocytes. With SBA and MAC, we could only remove 1.5 log, but with a
combination of OKT-C, SBA, and MAC, we eliminated 3.9 logs of clonogenic T
cells. Recoveries of hematopoietic progenitors were generally in excess of 50%.
We also developed a limiting dilution analysis for eliminating K562 cells
from peripheral blood or marrow samples (Christopher L. Reading et ai,
unpublished data). This analysis yields a clonogenic frequency for K562 cells of
nearly 1.0. We examined the ability of a combination of MAbs CF-1 and PM-81