Autologous Bone Marrow Transplantation - Blog Science Connections

570 Tumor Cell Detection
MATERIAL AND METHODS
Patient Population
Thirty-four patients at different stages of documented SCLC formed the
basis of the study for the human tumor cloning system. Thirty-nine patients at
the time of initial staging provided samples for the immunodetection. Staging
was exhaustive and aggressive. Fifteen normal bone marrow samples were
obtained from six healthy donors, one patient with a Ewing's sarcoma and
four with neuroblastoma.
Collection of Bone Marrow for Culture and Immunodetection
Bone marrow aspirates were collected in a syringe containing 125 0/ml
of Calparine (Choay, Paris, France). The samples were then diluted with an
equal volume of calcium- and magnesium-free Hanks' medium. Mononuclear
cells were concentrated by density centrifugation on Ficoll-Hypaque (1077
g/cm 3 ) (Pharmacia, Uppsala, Sweden) at 400 g on the interface during 30
minutes at 16° C. Next, the cell layer at the interface was collected, washed in
Hanks' medium, and centrifuged at 400 g for 20 minutes. Finally, the pellet
was counted using a Coulter Counter (Coulter Electronics 2 TD, England)
and resuspended in calcium- and magnesium-free phosphate-buffered saline
medium.
Human Tumor Cloning System
Cells were cultured according to Hamburger and Salmon (8), as
described by Pollard et at (9).
Anti-SCLC Rat Monoclonal Antibodies
The isolation and characterization of the MAbs anti-LCAl, LCA2, and
LCA3 have been described elsewhere (10). The antibodies were biotinylated
as described previously (11).
Indirect Immunofluorescence Assays
This assay was performed using each of the three MAbs separately. Cells
from the SCLC cell line, 250,000, or 500,000 isolated marrow cells were
incubated at 4°C during 30 minutes with 25 u\ of biotinylated MAb diluted to
1:100. After two washings with phosphate-buffered saline-fetal bovine serum
2%-Azide 5%, 50 u\ of fluorescent Avidine (Beckton Dickinson, California)
diluted to 1:100 was added to the pellet. Following an incubation period of 60
minutes at 4°C, the cells were washed two more times. After having mounted
cytocentrifuged cell smears in Tris-buffered glycerol (pH 9), the slides were
examined with a reflected light fluorescent microscope equipped with a 40 X
objective (Olympus Optical Co., Japan). The entire smear was scanned and
scored positive if at least one cell with bright surface fluorescence was
detected. Positive controls using the NCI-H69 line were made with each test.