autologous blood and marrow transplantation - Blog Science ...

5X10 5
Janssen et ai. 585
In brief, to measure the fraction of cells that were positive for the CD34 antigen,
cells to be assayed were suspended in 0.1 mL Dulbecco's phosphate
buffered saline (DPBS) (Gibco, Grand Island, NY) and admixed with 20 uL
phycoerythrin-tagged anti-CD34 (HPCA-2; Becton Dickinson, San Jose, CA).
After 30 minutes of incubation at 4°C, the cells were thrice washed with cold
DPBS and resuspended in 0.1 mL. The cell suspension was placed on a microscope
slide, and CD34-positive cells were scored by eye using fluorescence microscopy.
To measure the presence of CFU-GM, 2X10 5
cells were plated in 35-mm petri
dishes containing 1 mL Iscove's medium as well as 0.8% methylcellulose (Dow
Chemical, Midland, MI) or, in later studies, 1.15% methylcellulose (Stem Cell
Technologies, Vancouver, British Columbia) and supplemented with 20% fetal
bovine serum and 1 ng granulocyte-macrophage colony-stimulating factor (GM-
CSF) (Genzyme, Boston, MA). After 14 days of incubation, colonies of >25 cells
were scored.
Study design and statistical methods
To demonstrate that one stem cell product was superior to the other by
producing a one-third reduction of neutrophil and platelet recovery times with 80%
power at the P0.35.
All laboratory and patient data for these studies were maintained in computer
database files. In comparing days to engraftment, the log-rank test was employed.
Graphic representation of engraftment was produced using the Kaplan-Meier method
for computing probabilities of events. Comparisons of cell content returned to the
patients were carried out using the Kruskal-Wallis nonparametric analysis of variance
(ANOVA) method, as it was apparent that the numbers of cells collected were not
normally distributed. All statistical tests were carried out using Statistica software
(StatSoft, Tulsa, OK), and all values of P were computed from two-tailed tests.
RESULTS
Cells collected and infused
Seventy patients gave informed consent and had both BM and PBSC collected
after treatment with G-CSF. BM harvests were considered adequate if more than
2X10 8
mononuclear cells/kg were collected, and if the BM had a CD34 +
concentration of at least 0.5% (more than 10 6
/kg). PBSC harvests were considered
adequate if, after four collections, at least 10 6
CD34 +
cell
cells/kg had been collected.