Autologous Bone Marrow Transplantation - Blog Science Connections

Merocyanine 540 Marrow Decontamination 153
Table 1. Cloning Efficiency of Two Cell Lines After Mafosfamide and
Merocyanine 540 Treatment
Residual
Leukemia Cells (%) Treatment Blasts/Well (%)
CCRF-SB
5 AZ 0 22
5 MC-540 0 13.4
15 AZ 0.6 9.8
15 AZ +MC-540 0.25 7.3
K562
5 AZ 0.08 44
5 MC-540 0.11 40.3
15 AZ 1.75 1
15 AZ +MC-540 1.83 3.6
Note: Results were obtained from limiting dilution analysis of bone marrow mononuclear
cells contaminated with 5% or 15% of CCRF-SB lymphoblasts or myelogenous
K562 cells and subsequently treated with mafosfamide, merocyanine 540, or both. The
wells containing 10E4 purged mixture cells and to which clonogenic cells of the same
line were added using the limiting dilution technique were scored as positive
(responders) or negative (nonresponders) according to whether the tritium-labeled
thymidine uptake was greater or less than the threshold of the mean of the
nonresponding microculture with no added clonogenic cells plus three times the
standard deviation. Results were obtained by plotting the proportion of nonresponding
wells against the number of clonogenic cells added to each well (see Fig 1).
Abbreviations: CE, cloning efficiency; AZ, mafosfamide; MC-540, merocyanine 540.
CE
been described elsewhere (14,15). Table 1 and Figure 1 present the data
obtained from LDAs of cleansed HBMMC and leukemic cell mixtures in which
the equations of the line of best fit were derived using the maximum likelihood
method. As shown in Table 1, the cloning efficiencies (CEs) of CCRF-SB and
K562 cells, calculated as the slope of the regression line when the ordinate is the
natural log of the percentage of nonresponders, were 22% and 44%, respectively,
in the mafosfamide experiments, and 13.4% and 40.3%, respectively, in the
MC-540 experiments. When the clonogenic cell contamination was increased
by a factor of 3, the CE decreased in the mafosfamide studies and was not
improved by incubating the cell mixtures with both agents.
The estimate of the number of leukemic cells still present in the cell
mixtures after cleansing is represented by the x intercept of the maximum
likelihood regression line. In the experiments shown in Table 1, complete
decontamination of CCRF-SB cells in each well containing a mafosfamide- or
MC-540-purged mixture of 10 4
cells was obtained, but leukemic cells survived
after treatment. When the clonogenic cell frequency in the cell mixtures was
increased to 15%, CCRF-SB and K562 cells survived the treatment.
Incubation of murine marrow cells with 100 ug/ml of mafosfamide caused
a decrease in day 11 CFCJ-S colonies of about 50% of controls, and after