Paediatric diseases

In order to eliminate potentially CD34-positive tumor cells from autologous grafts, we established a clinical scale AC133 (CD133) selection procedure. Purity of AC133+CD34+ cells and contamination with residual neuroblastoma cells were evaluated. Fresh peripheral blood stem cell apheresis products (PBSC) with .20x10ˆ6/kg CD34+ from 2 pediatric patients with neuroblastoma and 6 cryopreserved PBSC cells were equally aliquoted and incubated with either magnetic bead conjugated AC133 or CD34 (QBEND 10) antibodies. Positive cells were selected using the Miltenyi SuperMACS or CliniMACS device. The 6 cryopreserved PBSCs of previously diseased patients were experimentally contaminated with a neuroblastoma cell line. Flow cytometric assessment of the selection product was on a EPICS XL-MCL (Beckman Coulter). Residual neuroblastoma cells in the PBSC and in the purified progenitor cells were determined by nested RT-PCR as well as by quantitative real time PCR for tyrosine hydroxylase and by immunocytochemistry. Purity of freshly isolated PBSC was 98.0% after AC133 selection and 97.3% after CD34 selection in accordance with the mean CD34+ purity of 96.8±4.2% in 71 previous CD34 selections in our laboratory. AC133 selection of cryopreserved PBSC achieved a purity of 91.8±8.0% (n=6) and CD34 selection led to a purity of 87.5±7.6% (n=6). Neuroblastoma cells were detected in PBSC of one patient and in all experimentally contaminated cryopreserved PBSCs prior to but not after AC133 nor CD34 selection. Large scale positive AC133 selection is feasible and achieves equivalent high purity of progenitor cells as previously reported for CD34 selection with effective tumor depletion. Therefore, AC133 purification may be a superior option to purge autologous grafts in patients with potentially CD34 positive tumor cells. Supported by ”Hilfe für Krebskranke Kinder Frankfurt e.V.”