Biomedical subjects
C Rouzier
Publications and source records attributed to C Rouzier.
Ex vivo expansion of CD34+/CD41+ late progenitors from enriched peripheral blood CD34+ cells.
In our experience, patients with neuroblastoma who undergo transplantation with CD34+ cells following high-dose chemotherapy have prolonged delays in platelet recovery. In vitro expansion of megakaryocyte (MK) cells may provide a complementary transplant product able to enhance platelet production in the recipient. We investigated the ability of a combination of various hematopoietic growth factors to generate ex vivo MK progenitors. Immunoselected CD34+ cells from peripheral blood stems cells (PBSCs) were cultured in media with or without serum, supplemented by IL-3, IL-6, IL-11, SCF, TPO, Flt-3 ligand, and MIP-1alpha. In terms of MK phenotypes, we observed a maximal expansion of CD61+, CD41+, and CD42a of 69-, 60-, and 69-fold, respectively, i.e., 8-10 times greater than the expansion of total cell numbers. Whereas the absolute increment of CD34+ cells was slightly elevated (fourfold) we showed increases of 163-, 212-, and 128-fold for CD34+/CD61+, CD34+/CD41+, and CD34+/CD42a+ cells, respectively. We obtained only a modest expansion of CFU-MKs after only 4 days of culture (fourfold) and similar levels of CFU-MKs were observed after 7 days (fivefold). Morphology and immunohistochemistry CD41+ analyses confirmed expansion of a majority of CD41+ immature cells on days 4 and 7, while on day 10 mature cells began to appear. These results show that primarily MK progenitors are expanded after 4 days of culture, whereas MK precursor expansion occurs after 7 days. When we compared the two culture media (with and without serum) we observed that increases of all specific phenotypes of the MK lineage were more elevated in serum-free culture than in medium with serum. This difference was especially marked for CD34+/CD61+ and CD34+/CD41+ (163 vs 42 and 212 vs 36, respectively). We contaminated CD34+ cells with a neuroblastoma cell line and we observed no expansion of malignant cells in our culture conditions (RT-PCR for tyrosine hydroxylase positive at day 4 and negative at day 7). With our combination of hematopoietic growth factors we are able to sufficiently expand ex vivo MK late progenitor cells to be used as complementary transplant products in neuroblastoma patients who undergo transplantation with CD34+ cells. It is possible that these committed MK late progenitors could accelerate short-term platelet recovery in the recipient until more primitive progenitor cells have had time to engraft.
Large-volume leukapheresis procedure for peripheral blood progenitor cell collection in children weighing 15 kg or less: efficacy and safety evaluation.
BACKGROUND: We update our experience on large-volume leukapheresis (LVL) in very small patients with malignancies. LVLs were performed with the aim of reducing the psychological impact of leukaphereses by reducing the number of procedures while collecting large numbers of cells. PROCEDURE: Seventeen LVLs were performed using a Cobe Spectra separator in 14 patients weighing < or = 15 kg. A median of 3.8 patient's blood volumes corresponding to 296 mL/kg (range, 202-565) of blood was processed per session of 190 minutes (120-279) duration. A femoral catheter was installed specially for collection for 88% LVL (vs. 35% for standard leukaphereses). A median volume of 16.9 mL/kg was collected with 5.4 x 10(8) MNC/kg (range, 0.6-16.3) and 8.2 x 10(6) CD34+ cells/kg (range, 1.3-31.7). RESULTS: No signs of complications due to citrate toxicity were encountered. No hypotensive or hypothermic episodes were observed. Platelet counts were significantly diminished after each procedure (median: -59%). When the extracorporal line was not primed with red blood cells (RBC), the difference between pre-LVL and post-LVL hemoglobin levels was significant with a median 32 g/L decrease. CONCLUSIONS: The LVL approach for peripheral blood progenitor cells (PBPC) collection in very small children may expose them to the risk of anemia and thrombocytopenia and an excess of special central line installation. The application of this technique in these patients should be reserved for special cases when a very large number of cells must be collected and should be performed by an experienced team.