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Background and Objectives: 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., Design and Methods: 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., Results: 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., Interpretation and Conclusions: 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.

Between February 1995 and August 1997, 11 children (eight males, three females) aged 4-16 years (median 7 years) underwent allogeneic PBPC transplantation for treatment of hematological disorders. Seven patients with acute leukemia (n = 5 ALL, n = 1 AML) or lymphoma (n = 1) received primary allogeneic PBPC transplantation, four patients received a second allotransplantation for graft failure (n = 1 AML, n = 1 sickle cell anemia) or disease recurrence (n = 1 ALL, n = 1 MDS). Five donors were HLA-identical siblings, five were 0-1 antigen mismatched family members and one was a matched unrelated donor. Donors received G-CSF 10-12 microg/kg/day for 3-7 days, and underwent one or two leukaphereses. The median cell yield per donor expressed per kg of recipient body weight was as follows: mononucleated cells 10.8 x 10(8)/kg (range 4.7-21.2); CD34+ cells 8.6 x 10(6)/kg (range 3.2-22); CD3+ cells 3.7 x 10(8)/kg (range 2.7-7.5). All patients achieved an ANC >0.5 x 10(9)/l after a median of 12 days (11-18). An unsupported platelet count >50 x 10(9)/l was reached 15 days (13-21) after PBPC transplantation; four patients failed to reach this threshold. Acute GVHD (aGVHD) grades II to IV occurred in eight (73%) patients: seven of them experienced grade III-IV aGVHD. Seven patients evaluable for chronic GVHD (cGVHD) were scored as absent in five, limited in one and extensive in one patient. As of September 1997, six patients (55 %) were alive between 60 and 938 days post-transplant (median follow-up 274 days); four patients with malignancy were alive in CR after primary allotransplantation, two patients were alive after a second PBPC transplant. Five patients have died with the main causes of death being aGVHD (n = 3), ARDS (n = 1), relapse of the underlying disease (n = 1). In conclusion, despite the limited number of patients, these preliminary results indicate that PBPC may be considered as an alternative to bone marrow for allografting also in children.

Preliminary BM processing to produce an enriched MNC fraction from large BM volumes improves subsequent pharmacological and/or immunological "ex vivo" treatment and cryopreservation. We detail on a multicenter study (6 Transplant Centers) performed to establish an effective and reliable protocol using a CS 3000 continuous flow separator on a large series of BM processed for autologous (96) and allogeneic (12) transplantation. The reduction in volume was 78.6 + 7.2% while 28.9 + 12.4% of the original nucleated cells were found in the final product. A mean of 84.3 + 13.2% of the staring MNC was yielded in a fraction containing over 81% MNC. Cloning efficiency indicated than the final graft was highly enriched in progenitor cells committed to the granulocyte/macrophage pathway (> 100%) as assessed in vitro (CFU-GM). Removal of RBC and PLT was 98.3 + 1.1 and 37.7 + 14.6%, respectively. The mean dose of MNC and CFU-GM was 0.6 + 0.37 x 10(8) and 0.96 + 1 x 10(5) recipient weight. The entire process was accomplished in 87.5 + 20 min. We concluded that this automated device is a simple and reproducible method for BM processing suitable as first step for further "ex vivo" automated negative and/or positive cell selections.

The functional capacity of T lymphocytes from 28 cases of chronic T-cell leukaemia--T-CLL, T-PLL, T-LCL and Sézary syndrome--was evaluated in a T-colony forming system and in a PHA response assay. Reduced or absent T-colony growth was observed in 23 cases (82%) while in five the growth was normal. Although a good correlation was generally observed between colony formation and PHA transformation, in a few cases a low PHA response was accompanied by moderate colony growth and vice versa. Characterization of the leukaemic T lymphocytes using monoclonal antibodies (OKT series) indicates that cases with a helper/inducer phenotype (OKT4+) showed moderately reduced or near-normal T-colony numbers, whilst cases with a suppressor/cytotoxic phenotype (OKT8+)--confined to T-CLL in this study--had a very low or absent colony growth. The functional abnormalities reported here suggest that neoplastic T-cells with a helper/inducer phenotype show a low proliferative response in the assay systems used, although expressing mature T-cell characteristics. The low growth observed in T-CLL confirms that cells with a suppressor/cytotoxic phenotype form few T-cell colonies.

The distribution of T cell subsets and the T colony-forming capacity were assessed in 22 patients with hairy-cell leukemia. An overall increase in the proportion of T gamma cells (31 +/- 15%) and a decrease in Tmu cells (38 +/- 11%) were observed if compared with normal controls (16 +/- 6% for T gamma and 51 +/- 13% for Tmu). In half of the patients, however, these values were normal. T gamma cells were highest in the splenectomized patients and in the 15 non-splenectomized patients T gamma cells were higher in those with active disease. Patients with stable disease or in remission had values close to normal. The T colony-forming capacity of unseparated and enriched T lymphocytes was normal regardless of the T cell subset distribution and clinical stage of the disease. These findings suggest that in HCL, unlike B cell chronic lymphocytic leukemia, the imbalance in Tmu/T gamma ratio does not result in an impairment of the in vitro T colony-forming capacity.

Four children with measles meningoencephalitis were treated with thymostimulin, a calf thymic extract. All patients were comatose when therapy was started. In all but one a good and sometimes rapid clinical improvement was observed. In the responsive patients no neurological or EEG sequellae were noted over a follow-up period of 1-4 years. The depression of cell-mediated immunity, present in some patients before treatment, normalised at the end of therapy.

Maintaining a high haemoglobin level, through a high transfusion regime, is the best method for treating thalassaemia. Not much is known about the effect of this treatment on medullary or extramedullary haemopoiesis, particularly on the extent of erythropoietic inhibition and on the behaviour of myelopoiesis. In order to analyse some aspects of the problem, we studied the myeloid stem cells (CFU-c) in the bone marrow and in the peripheral blood of children with homozygous thalassaemia, using the agar culture technique. The number of circulating CFU-c observed in 68 patients was higher than in normal subjects. This number was significantly increased after splenectomy. A positive correlation was demonstrated between the number of circulating CFU-c and the time elapsed since the last transfusion. Patients with a high Hb level displayed a marked reduction in the number of CFU-c in their peripheral blood. In 10 patients, before the beginning of transfusions, bone marrow CFU-c were lower than in normal subjects; their number increased after therapy. Most circulating CFU-c were proliferating as shown by the thymidine suicide technique.

Hairy cell leukaemia (HCL) cells on exposure to the tumour promoter tetradecanoyl-12,13-phorbol acetate (TPA) undergo a striking morphological change in culture, with the formation of long, slender cytoplasmic processes, and adhere to the plastic surface. By time lapse photography this change is seen to start within 10 min of exposure to the TPA and is characterised by slow but continuous extension and retraction of the processes. Apparent phagocytosis of sheep red cells but not of sensitised human red cells occurs. This response to TPA is prevented by cytochalasin D but not by 13 cis-retinoic acid. Lymphocytes from normal individuals and patients with chronic lymphocytic leukaemia and lymphoblasts from a patient with acute lymphoblastic leukaemia reacted by adhering to each other in small aggregates but did not adhere to the plastic surface. Monocytes adhere to plastic surfaces and respond to TPA by extension and retraction of cytoplasmic folds differing from the long slender processes formed by the HCL cells. Thus HCL cells are different in their response to TPA from both normal lymphocytes and monocytes.