[Quality control of defrosted cord blood units: results from an inter-laboratory study]. / Contrôle de qualité des greffons de sang placentaire décongelés : résultats d'une étude multicentrique.

PURPOSE: Today, haematopoietic stem cell graft from placental blood concerns more than 15 % of allogeneic grafts. An inter-laboratory study of the quality control of defrosted cord blood units has been coordinated by the French society for cell and tissue bioengineering (SFBCT), with the cord blood bank of Bourgogne Franche-Comté and controlled by the French health products safety agency (Afssaps). The aim of this study is to ensure the inter-laboratory reproducibility of the quality controls practised by the banks during defrosting. The cellular outputs were analyzed according to the defrosting techniques, according to the method used in flow cytometry: single-platform (SP) versus double-platform (DP), or the product nature, i.e. in total blood or miniaturized. METHODS: Forty-two units of placental blood (USP), which were out of range were provided for defrosting to 14 participating sites. USP were defrosted and controlled according to the procedures of each bank. Once the USP is defrosted, a part of the product was controlled by the site and the other part by Afssaps. Following controls were carried out: numeration of the total nucleated cells (TNC) and of CD34+ cells (made by a SP method in Afssaps) and functional assay. RESULTS: Concerning TNC, the defrosting sites obtained a cellular output of 94 %+/-28 in day 0 compared with an output of 72 %+/-24 in Afssaps showing a rather good stability of the USP transmitted with an average deviation of 23 %+/-22. The freezing process with or without reduction of volume does not affect this variation. Concerning the numeration of CD34+ cells, the average deviation between the participating sites and Afssaps was 29 %+/-23 compared with 21 %+/-16 for the sites using a SP method against 47 %+/-25 for those using a DP method. The CD34+ outputs are equal to 82 % +/- 60 in day 0 for the participating sites against 52 %+/-20 for Afssaps. For the sites using a DP method, it is stressed that this output is particularly high with a rate of 126 %+/-90 (n=15) whereas it is 62 %+/-20 (n=32) for the sites using a SP method. CONCLUSION: These results underline a good stability of viable CD34+ cells and a greater reliability of the SP methods for the CD34+ cell numeration for these defrosted USP. Lastly, the results of the functional assay regarding the average clonogenicities (equal to 15 %) reinforce the conclusions on the quality of the defrosted products.

Uncontrolled-rate freezing and storage at -80 degrees C, with only 3.5-percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations.

BACKGROUND: Although controlled-rate freezing and storage in liquid nitrogen are the standard procedure for peripheral blood progenitor cell (PBPC) cryopreservation, uncontrolled-rate freezing and storage at -80 degrees C have been reported. STUDY DESIGN AND METHODS: The prospective evaluation of 109 autologous PBPC transplantations after uncontrolled-rate freezing and storage at -80 degrees C of apheresis products is reported. The cryoprotectant solution contained final concentrations of 1-percent human serum albumin, 2.5-percent hydroxyethyl starch, and 3.5-percent DMSO. RESULTS: With in vitro assays, the median recoveries of nucleated cells (NCs), CD34+ cells, CFU-GM, and BFU-E were 60.8 percent (range, 11.2-107.1%), 79.6 percent (6.3-158.1%), 35.6 percent (0.3-149.5%), and 32.6 percent (1.7-151.1%), respectively. The median length of storage was 7 weeks (range, 1-98). The median cell dose, per kg of body weight, given to patients after the preparative regimen was 6.34 x 10(8) NCs (range, 0.02-38.3), 3.77 x 10(6) CD34+ cells (0.23-58.5), and 66.04 x 10(4) CFU-GM (1.38-405.7). The median time to reach 0.5 x 10(9) granulocytes per L, 20 x 10(9) platelets per L, and 50 x 10(9) reticulocytes per L was 11 (range, 0-37), 11 (0-129), and 17 (0-200) days, respectively. Hematopoietic reconstitution did not differ in patients undergoing myeloablative or nonmyeloablative conditioning regimens before transplantation. CONCLUSION: This simple and less expensive cryopreservation procedure can produce successful engraftment, comparable to that obtained with the standard storage procedure.

Granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells from children contain the same levels of long-term culture-initiating cells producing the same numbers of colony-forming cells as those from adults, but display greater in vitro monocyte/macrophage potential.

Autologous peripheral blood progenitor cell (PBPC) transplantation is now commonly used in children. The ontogenic differences in haematopoiesis published in recent years suggest differences in the categories of mobilized PBPCs between children and adults. We investigated the frequency and distribution of mature progenitor cells (colony-forming cells, CFCs) and primitive progenitor cells [CD34+ CD38- and CD34+ Thy-1+ cells, long-term culture-initiating cells (LTC-ICs)] in children and adults mobilized using granulocyte colony-stimulating factor alone. We found similar proportions of granulocyte colony-forming units (CFU-G) and/or macrophage CFUs (CFU-M), mixed lineage CFUs (CFU-Mix) and megakarocyte CFUs (CFU-Mk), CD34+ CD38- and CD34+ Thy-1+ cells, and LTC-ICs (16.5 +/- 3.5 vs. 10.65 +/- 5 per 104 CD34+ cells), which produced the same number of CFCs (5 +/- 1 vs. 6 +/- 1 CFCs/LTC-ICs) in PB CD34+ cells from children and adults. However, we noted a higher proportion of erythroid blast-forming units (BFU-E) in PB CD34+ cells from adults (x 1.5, P = 0.003). Using cord blood as a third ageing point, we observed an inverse age-related propensity for commitment to the monocyte/macrophage lineage that was still found after normalizing the data per body weight and processed blood mass. This ontogeny-related programming was detected from the LTC-IC level, which produced 1.7 times more CFU-M in children than in adults (P = 0.048). These subtle differences in commitment between children and adults, shown here for the first time, are of interest for the in vitro manipulation of PBPCs and, in particular, for application in adoptive immunotherapy in children.

Granulocyte colony-stimulating factor alone at 20 micrograms/kg vs. 10 micrograms/kg for peripheral blood stem cell mobilization in children.

Mobilization of peripheral blood stem cells (PBSC) by granulocyte colony-stimulating factor (G-CSF), at 10 micrograms/kg/day vs. 20 micrograms/kg/day (in 42 and 29 patients, respectively), was compared in children with solid tumors or leukemias. During mobilization, differences were noted in the peak values of CD34+ cells in peripheral blood (PB) in these two groups (median 28 x 10(6)/L for 10 micrograms/kg/day vs. 61 x 10(6)/L for 20 micrograms/kg/day; p = 0.025). Similar numbers of progenitor cells were harvested for the two concentrations of G-CSF. However, similar CD34+ cell levels in the leukapheresis product were obtained after only the third dose of G-CSF at 20 micrograms/kg/day compared with the fourth dose of G-CSF at 10 micrograms/kg/day (1.7 and 1.2 x 10(6) CD34+ cells/kg/one patient's blood volume processed, respectively). Of note is the impact of diagnosis on PB CD34+ cell levels. We conclude that, in children, mobilization with G-CSF at 20 micrograms/kg/day could minimize the duration of priming but not reduce the number of leukaphereses. Thus, the impact on outcome, clinical practice, bed utilization, and health economics is uncertain.

Two-step immunoablative treatment with autologous peripheral blood CD34(+) cell transplantation in an 8-year-old boy with autoimmune haemolytic anaemia.

Life-threatening haemolysis in children with autoimmune haemolytic anaemia (AIHA) occurs rarely. Many cases of severe autoimmune disease are currently treated with immunosuppressive high-dose chemotherapy and autograft. We report here a case of a child with severe AIHA who did not respond to conventional treatments, but was cured with an autologous peripheral blood CD34(+) cell transplantation. After d 16 post autograft, no further red cell transfusions were required. At 20 months post autograft, haematological complete remission persists.

CFU-Mk content of immunoselected CD34+ peripheral blood progenitor cells, evaluated with an adapted serum-free methylcellulose assay, is predictive of platelet lineage reconstitution in children with solid tumors.

Immunoselected CD34+ peripheral blood progenitor cell (PBPC) transplantation is now frequently used to support autologous hematopoiesis after myeloablative therapy, its feasability having been proved by several groups. However, we and others observed delayed platelet recovery. We hypothesized that immunoselection processing might induce selective loss of megakaryocyte progenitors, or a decrease in their proliferation. We used a colony-forming units megakaryocyte (CFU-Mk) assay to evaluate these consequences and predict platelet recovery in patients. In CD34+ PBPCs from 10 children with solid tumors, we observed no selective loss in CFU-Mk numbers during immunoselection processing and no impairment of clonogenicity. The CFU-Mk yield (59.2 +/- 11.3%) was at least similar to the CD34+ yield (44.2 +/- 3.8%). We assessed the predictive value of CFU-Mk numbers infused for recovery of platelet lineage. We found an inverse correlation between the time taken to reach a platelet count greater than 50 x 10(9)/L and only the CFU-Mk dose (r = -0.71; p = 0.022) among the different type of progenitors, including colony-forming units granulocyte-macrophage (CFU-GM), burst-forming units erythrocyte (BFU-E) and colony-forming units-mixed (CFU-Mix). These findings suggest that CFU-Mk number could be used as sole predictive functional parameter for platelet reconstitution in children after immunoselection of CD34+ cells, in particular for low CD34+ cell dose, and thus as an indicator for initial quality of hematopoietic cells before in vitro expansion.

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.

Peripheral progenitor cells (CFU-GM, BFU-E, CD34) are increased in untreated chronic lymphocyte leukemia patients: stage B and C display a particularly high CD34+ cell count.

No treatment has proved its efficiency in CLL. Autologous transplantation is now under consideration for the youngest patients. We assayed progenitor cells (CFU-GM, BFU-E, CD34) in the peripheral blood of 28 untreated CLL patients and found an increase of all these progenitors in CLL compared to controls. There was no statistical difference between stage A versus stages B and C for CFU-GM and BFU-E. In contrast, CD34 cells were higher in stages B and C as compared to stage A. This finding could be explained by a high number of circulating clonal cells in advanced stages of the disease.

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.

Kinetics of hematopoietic progenitor cell release induced by G-CSF-alone in children with solid tumors and leukemias.

The kinetics of peripheral blood progenitor cell (PBPC) release induced by G-CSF-alone at 10 microg/kg/day were monitored daily in 42 children with solid tumors and leukemias. Median 16- and 27-fold enrichment of circulating CD34+ cells and granulocyte-macrophage colony-forming units (CFU-GM) was noted with peak values occurring after the 4th or the 5th G-CSF dose. Individual values of PBCD34+ cell levels in patients with solid tumors were not significantly different after the 4th and after the 5th dose. The day-of-collection PBCD34+ cell concentration was related to the harvested CD34+ cell (P = 0.0001) and CFU-GM numbers (P = 0.0001). No correlations were found between PBPC enrichment and either patient age, body weight, diagnosis or pre-mobilization treatment duration. The median numbers of 1.1 x 10(6) CD34+ cells/kg and 28.1 x 10(4) CFU-GM/kg were derived from one patient's blood volume processed. Nineteen patients received G-CSF-alone primed grafts and had successful engraftment. Our data indicate that in 88% of children a single standard leukapheresis is sufficient to obtain a minimum graft (2 x 10(6) CD34+ cell and/or 10 x 10(4) CGU-GM per kg) whether undertaken after the 4th dose of G-CSF or the 5th.

Molecular monitoring of tumor cell contamination in leukapheresis products from stage IV neuroblastoma patients before and after positive CD34 selection.

BACKGROUND: Autologous peripheral blood stem cell (PBSCs) are frequently used to reconstitute hematopoiesis following administration of megatherapy in children with advanced stage IV neuroblastoma. Some centers prefer the use of autografts enriched for CD34+ progenitor cells because the positive selection procedure is believed to reduce indirectly tumor cell contamination. PROCEDURES: In this study, we monitored the efficiency of tumor cell purging following CD34 selection in PBSCs from seven patients with advanced neuroblastoma by using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, Amplification of tissues-specific mRNA transcript of tyrosine hydroxylase gene with nested primers enabled the detection of residual neuroblastoma cells with a sensitivity of one malignant-cell per 10(6) normals. RESULTS: Using this method, contaminating tumor cells were detected in seven of nine leukapheresis products of the patients. After positive immunoselection of CD34+ cells on Ceprate column, only one of nine enriched stem cell fraction still contained tumor cells detectable by RT-PCR. In six cases, PCR positive PBSCs became PCR negative after selection. CONCLUSIONS: We conclude that tumor cell contamination may be frequently detected in PBSC harvests of stage IV neuroblastoma patients by sensitive molecular analysis. The load of contaminating malignant cells might be reduced following CD34 selection.

Safe and efficient peripheral blood stem cell collection in the smallest of children.

To clarify the factors that may affect the peripheral blood stem cell (PBSC) collection in children weighing < or = 15 kg, a consecutive registry of 109 leukapheresis procedures was analyzed. Collections were performed on a COBE Spectra separator. In 65.1% of the procedures, the peripheral vein, together with a central catheter inserted routinely at diagnosis, or 2 peripheral veins were used to access/return. For 84.4% of the procedures, the extracorporeal line was primed with red blood cells. The median granulocyte-macrophage colony forming unit (CFU-GM) number derived from 1 patient's blood volume processed was 13.8 x 10(4)/kg. Six times, a collection series failed, always in children treated for > or = 26 weeks and 4 of those times in children weighing < or = 11 kg. The patient's age, diagnosis, duration of preleukapheresis treatment, and mobilization regimens did not significantly affect the collection yield. Twenty-four transplantations were performed. The median times to neutrophils >0.5 x 10(9)/L and platelets >20 x 10(9)/L were 13 and 20 days, respectively. We conclude that even in very small children, leukapheresis can be performed safely, allowing adequate PBSC collection for transplantation and/or in vitro manipulations.

Feasibility of a PB CD34+ cell transplantation procedure using standard leukapheresis products in very small children.

To evaluate the feasibility and efficacy of CD34+ cell immunoselection from routine peripheral blood stem cell (PBSC) harvests in very small children a prospective study was performed in 15 children with advanced neuroblastoma weighing 20 kg or less. Products of two consecutive leukaphereses carried out on a COBE Spectra separator after G-CSF alone mobilization were pooled for immunoselection on Ceprate column. The median number of CD34+ cells and total CFU-GM collected were respectively 5.9 x 10(6)/kg (range 2.3-23.4) and 126.9 x 10(4)/kg (range 52.9-559.9). After separation the median number of CD34+ cells in the adsorbed fraction was 2.6 x 10(6)/kg (range 1-9.8) with a median purity of 54% (range 21-82) and a median of 95.7-fold (range 35-250) enrichment. Thirteen patients underwent autografts with CD34+ PBSCs after a busulfan 600 mg/m2 + melphalan 180 mg/m2 preparative regimen. The median number of days to achieve an absolute granulocyte count of 0.5 x 10(9)/l and a platelet count of 20 x 10(9)/l were respectively, 12 (range 10-24) and 35 (range 25-43). The median number of platelet transfusions was nine (range 2-15). We conclude that safe and effective immunoselection and transplantation of CD34+ PBSC can be accomplished in children with low body mass.

CD34+ cell immunoselection from G-CSF-alone-primed peripheral blood in children with low body mass.

We report the data of CD34+ cell immunoselection from peripheral blood after G-CSF-alone mobilization (10 micrograms/kg/d s.c.) in nine children with neuroblastoma (median age 4-5 years (2-8), median body weight 16 kg (10-20). Leukaphereses were carried out on a Cobe Spectra separator and two consecutive harvests (4 blood volumes processed) were used for immunoselection on a Ceprate column. The yield of CD34+ cells in the purified fraction was 50% (23-80), with a median number of 2.8 x 10(6) CD34+ cells/kg (1-9.4). All patients were reinfused with selected CD34+ cells after busulfan 600 mg/m2 +melphalan 180 mg/m2 and achieved successful haemopoietic recovery.

Use of G-CSF alone to mobilize peripheral blood stem cells for collection from children.

We report the data of 19 children with neuroblastoma (NB) or Ewing's sarcoma (EW) who had peripheral blood stem cells (PBSCs) harvested after mobilization by: (1) cyclophosphamide (CY) + etoposide + G-CSF, (2) CY + GM-CSF, or (3) G-CSF alone. There were no consistent differences in the number of PBSCs collected following these three different mobilization regimens as assessed by CFU-GM. 17 patients were reinfused with PBSCs after myeloablative therapy and had successful haemopoietic recovery. These results show that in children with solid tumours such as NB or EW a sufficient number of PBSCs can be collected after G-CSF alone, and that PBSCs collected following stimulation by G-CSF alone are as effective in reconstituting haemopoiesis as those collected after mobilizing chemotherapy + HGFs.

[Cytogenetic study of 20 cases of refractory anemia]. / Etude cytogénétique de 20 cas d'anémies réfractaires.

The myelodysplastic syndromes are a group of hematological disorders not yet clearly defined. The authors describe the chromosomal aspects of 20 cases observed in the region of Clermont-Ferrand in comparison with the bibliographical data.