autologous blood and marrow transplantation - Blog Science ...

564 Chapter 10: Graft Manipulation
or 300i (one procedure) Magnetic Cell Separation Systems (Baxter
Immunotherapy). Products containing more than 0.5% CD34 +
cells and more than
5X10 6
CD34 +
cells/kg were chosen preferentially for selection. The enrichment
was performed on the day of leukapheresis, as in our hands overnight storage
resulted in a substantial loss of viable CD34 +
cells. If possible, uhmanipulated
leukapheresis products were cryopreserved as back-up.
After a platelet wash of 10 minutes at 250g, the cells were resuspended in 100
mL phosphate-buffered saline (PBS, calcium- and magnesium-free) containing 1%
human serum albumin (HSA) (Immuno, Heidelberg, Germany), 0.2% sodium
citrate (Braun, Melsungen, Germany), and 0.5% immunoglobulin (Venimmun,
Behring, Marburg, Germany). The cells were incubated with an anti-CD34
monoclonal antibody (Baxter) for 30 minutes at 4°C while rotating. After one
wash, 4X10 9
freshly prepared sheep anti-mouse IgGl (Fc) paramagnetic beads
(Dynabeads M450; Dynal, Oslo, Norway) were added to the cells in a final volume
of 200 mL. The suspension of cells and beads was incubated for 30 minutes at room
temperature while rotating. The complexes of beads and cells were retained by the
primary magnet of the Isolex 300SA, whereas unbound cells were removed by
three washes. Rosetted cells were released from the immunomagnetic beads after
incubation with chymopapain for 15 minutes at room temperature (two procedures)
(8000 pKAT, ChymoCell-T; Baxter) or using a competitor peptide (PR34; Baxter)
(30 procedures). After release, the beads were captured by repeated passages over
the magnet. After washing, CD34 +
cells were resuspended in freezing medium
containing minimal essential medium (MEM) (Seromed, Biochrom KG, Berlin,
Germany), 7.5% dimethylsulfoxide (DMSO) (Merck, Darmstadt, Germany), and
4% HSA in a final volume between 5 and 15 mL. The cell suspension was frozen
in 5 mL Nalgene tubes (Nalgene, Rochester, NY) using a computer-controlled
freezing device (CryolO-16 MR2; Messer-Griesheim, Germany). The tubes were
stored at - 196°C in the liquid phase of nitrogen.
Immunofluorescence staining and flow cytometry
For immunofluorescence analysis, 10 6
cells were incubated for 30 minutes at
4°C in the presence of an anti-CD34 fluorescein isothiocyanate (FITC)-conjugated
HPCA-2 and an anti-CD 19 phycoerythrin (PE)-conjugated monoclonal antibody
(all from Becton Dickinson, Heidelberg, Germany). Isotype identical antibodies
(Becton Dickinson) served as controls. Cells were washed twice with PBS, and red
blood cells were removed using a FACS lysis solution (Becton Dickinson).
Immunofluorescence analysis was performed using a five-parameter FACScan
(Becton Dickinson) equipped with an argon-ion laser tuned at 488 nm and 0.3 W.
Emission from FITC and PE was measured using filters of 530 nm and 585 nm,
respectively.