autologous blood and marrow transplantation - Blog Science ...

Mapara et al. 531
products of patients with nonhematological malignancies using the Isolex 300
device (Baxter Biotech, Munich, Germany) as previously described. 12
CD34 +
hematopoietic progenitor cells were cultured in Iscove's modified Dulbecco's
medium (IMDM) containing 20% fetal calf serum (FCS), interleukin (IL)-3 (100
U/mL), IL-6 (100 U/mL), and stem cell factor (50 mg/mL). All growth factors were
purchased from Promocell (Heidelberg, Germany). Patients were mobilized either
with granulocyte colony-stimulating factor (G-CSF) alone (12 ug/kg; Amgen,
Thousand Oaks, CA) or in combination with chemotherapy (epirubicin 12 mg/m 2
plus ifosfamide 7.5 g/m 2
) in established therapy protocols.
CD34 selection and subsequent immunomagnetic tumor cell depletion
Leukapheresis bags were stored in the original cell collection bag of the C4Y
collection set (Fresenius) up to 72 hours at 4°C in autologous plasma and ACD-A.
Bags stored for 24, 48, and 72 hours had a mean cell concentration of
65.5X 10 6
/mL (±26.14x 10 6
/mL) and 58X 10 6
/mL (±42.67X 10 6
/mL), respectively.
Since the Fresenius AS 104 cell separator was used for apheresis, cells are directly
collected in autologous plasma plus ACD-A in a total volume of 280-360 mL.
Trypan blue dye exclusion staining revealed a mean cellular viability of 100 ± 0.7,
99 ± 1, 99 ± 1.5% after 24, 48, and 72 hours, respectively. Median total CFU
formation per 2X10 4
mononuclear cells (MNC) was 202 ± 88, 247 ± 121 after 24,
48, and 72 hours, respectively. Median viability after the first platelet wash of
pooled LP was 98%.
Enrichment of CD34 cells from leukapheresis product (LP) was performed
using the ISOLEX 300SA and the ISOLEX 300i device (Baxter Biotech)
according to the protocol provided by the manufacturer. For the ISOLEX SA,
after washing (3X Dulbecco's phosphate-buffered saline [DPBS], 0.2% sodium
citrate [wt7vol], 1% human albumin at room temperature, 200g for 10 minutes)
cells were incubated with the anti-CD34 mAb 9C5 (0.5 ug/lX10 6
cells for 30
minutes at 4°C). Unbound antibody was removed by washing as mentioned
above. Thereafter, sensitized cells were incubated with immunomagnetic beads
(4.5 um) coated with goat anti-mouse antibodies (Cynal AS, Oslo, Norway).
Subsequently, cells were captured by a magnet in the ISOLEX 300 chamber.
Cells were released from the beads using the peptide release agent (PR34+).
After collection and washing of the CD34 +
fraction, cell count and viability
testing were performed. CD34 selection using the ISOLEX 3001 was performed
as follows: after storage of leukapheresis products, bags were pooled and a
platelet wash was performed. The volume was adjusted to 30 mL with working
buffer (PBS, 1% human albumin, 4.5% ACD-A [vol/vol]). Cells were incubated
for 15 minutes at room temperature with a human immunoglobulin preparation
(5% Gammagard; Baxter) before the enrichment procedure. After priming of the