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Cord blood (CB) is a valuable source of stem cells. Most CB units are still cryopreserved in single bags in the world's CB banks. Thawing a single CB unit, dividing it into two parts, expanding the smaller one and refreezing the other would optimize ex vivo expansion of CB progenitors prior to transplantation: expanded and unexpanded cells could be infused together to accelerate early engraftment.The feasibility of refreezing CB samples was investigated by evaluating the effect of 3 successive cryopreservation procedures in 9 CB units. The number and viability of WBC, BFU-E, CFU-GM, CFU-MIX, LTC-IC, and the absolute CD34+ cell count were assessed at time 0 and after each thawing. The percentage of CD34 cells expressing CD38, L-selectin, VLA-4, VLA-5, H-CAM, LFA-1 and CXCR4 was also evaluated.After three freezing and thawing procedures, WBC counts decreased, while lymphocytes were unchanged. Viability was 90% of basal values after the first thawing and did not change. BFU-E decreased significantly only after the third thawing. CFU-GM and CFU-MIX did not change significantly, nor did LTC-IC, CD34+ cell counts and CAM and CXCR4 expression on CD34+/ CD38-- cells.These data show that two successive freeze-thaw procedures do not significantly affect the clonogenic potential and CAM expression of cord blood progenitors. This information could be exploited to devise new options in ex vivo expansion procedures and quality controls prior to transplantation.

Granulocyte colony-stimulating factor (G-CSF) has been shown to improve the neutropenic status of patients with bone marrow failure. The side effects in prolonged treatment still need to be determined.We have studied the efficacy and the long-term side effects of G-CSF in four patients with Fanconi's anemia and severe neutropenia.Three patients responded with an increase in their absolute neutrophil count; neither improvement in platelet count and hemoglobin concentration nor effect on transfusion requirements was seen. CFU-GM and BFU-E were undetectable before, during and after treatment. Responders showed an important reduction in number and severity of infections, with a marked improvement of clinical status. The fourth patient developed acute myeloid leukemia after 4 weeks of G-CSF treatment. During maintenance, one patient was treated with G-CSF for 18 months, until she received bone marrow transplantation, without presenting side effects. In the second responding patient G-CSF treatment was stopped because of appearance of immature cells in peripheral blood and myeloid blasts in bone marrow. The third responding patient presented immature peripheral myeloid cells during the third year of G-CSF treatment: disappearance of immature cells was observed after G-CSF reduction. In two cases FISH analysis revealed monosomy 7 after G-CSF treatment.G-CSF use results in an improvement of clinical status, but long term administration may cause adverse experiences and requires a close hematological monitoring.