autologous blood and marrow transplantation - Blog Science ...

Ross et al.
post-TEC fraction was collected after release of the bound cells by manual
disruption of the column bed. Cytospin preparations of the entire pre-TEC fraction
for each sample were made. Cytospin preparations from the pre-TEC, post-TEC,
and FT fractions were immunostained with the cocktail of anti-cytokeratin mAbs
for the presence of tumor cells as detailed below.
A minimum of two experiments was performed for each seeding concentration.
For the following tables, these calculations were used to express tumor cell
recovery and enumeration:
' recovery of total tumor cells =
(number tumor cells in post-TEC fraction ) X 100%
number tumor cells seeded in pre-TEC sample
... . . % of tumor cells in post-TEC fraction
fold enrichment = — -—— (2)
% tumor cells in pre-TEC sample
log enrichment = log (fold enrichment) (e.g., log of 500 = 2.69) (3)
total tumor cells in TEC (FT) =
(tumor cell count) (volume of FT fraction)
mL
Patient specimens
Bone marrow and PBSC apheresis specimens were obtained from researchers at
collaborating institutions. Both fresh and cryopreserved specimens were analyzed.
Specimens were split, with 5.0X10 6
cells reserved for standard pre-TEC ICC
analysis. A minimum of 1.0X10 8
cells were incubated with the biotinylated TEC
mAb and processed as described above.
ICC staining
The pre-TEC, post-TEC, and TEC FT specimens from both tumor cell seeding
and patient specimens were prepared as cytospins and fixed in a mixture of
Histochoice (AMRESCO, Solon, OH) and 4% paraformaldehyde for 30 minutes at
4°C and rinsed thoroughly in phosphate-buffered saline (PBS). The slides were
then loaded on the TechMate 500 (Ventana Medical Systems, Tucson, AZ)
immunostainer and placed in blocking solution for 25 minutes. They were then
rinsed in PBS and incubated for 20 minutes in an anti-cytokeratin mAb cocktail.
The slides were then rinsed in PBS, incubated for 20 minutes in the secondary
antibody, and incubated for an additional 20 minutes in the ABC-alkaline
523
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