We have developed an avidin-biotin immunoadsorption technique in conjunction with a monoclonal anti-CD34 antibody that is capable of selecting CD34+ progenitor cells from marrow and mobilized peripheral blood. Clinical studies with these CD34+ selected cells have shown that the cells are capable of rapid and durable engraftment. In addition, there is significantly less infusional toxicity to the patient because the volume in which the CD34+ selected cells are contained is much less than that of a typical marrow or apheresis buffy coat. Selection of CD34+ progenitor cells also offers other potential advantages, including T-cell depletion of allografts and tumor cell depletion of autografts. CD34+ selection can also be used to facilitate other manipulations of marrow and peripheral blood, including gene transfection, ex vivo stem cell expansion, tumor purging, and progenitor cell banking. Future graft engineering studies are expected to clarify these relationships and enable refinement of the graft to the point at which GVHD can be minimized, graft survival maximized, and relapse-free survival prolonged.
Summary:The purpose of this study was to investigate whether storing mobilized peripheral blood progenitor cell (PBPC) collections overnight before CD34 ؉ selection may delay platelet count recovery after high-dose chemotherapy and CD34 ؉ -enriched PBPC re-infusion. Lymphoma patients underwent PBPC mobilization with cyclophosphamide 4 g/m 2 i.v. and G-CSF 10 g/kg/day subcutaneously. Patients were prospectively randomized to have each PBPC collection enriched for CD34 ؉ cells with the CellPro CEPRATE SC System either immediately or after overnight storage at 4؇C. Thirty-four patients were randomized to overnight storage and 34 to immediate processing of PBPC; 15 were excluded from analysis due to tumor progression or inadequate CD34 ؉ cell mobilization. PBPC from 23 patients were stored overnight, while 30 subjects underwent immediate CD34 ؉ selection and cryopreservation. Median yield of CD34 ؉ enrichment was 43.6% in the immediate processing group compared to 39.1% in the overnight storage group (P ؍ 0.339). Neutrophil recovery Ͼ500 × 10 9 /l occurred a median of 11 days (range 9-16 days) in the overnight storage group compared to 10.5 days (range 9-21 days) in the immediate processing group (P ؍ 0.421). Median day to platelet transfusion independence was 13 (range 7-43) days in the overnight storage group vs 13.5 (range 8-35) days in those assigned to immediate processing (P ؍ 0.933). We conclude that storage of PBPC overnight at 4؇C allows pooling of consecutive-day collections resulting in decreased costs and processing time without compromising neutrophil and platelet engraftment after infusion of CD34 ؉ -selected progenitor cells. Bone Marrow Transplantation (2000) 25, 559-566.
The kinetics of mobilization and optimal timing of peripheral blood progenitor cell (PBPC) collection were evaluated in 190 patients with multiple myeloma undergoing stem cell harvest after mobilization with cyclophosphamide, prednisone and G-CSF. There was a strong correlation between the WBC count and the number of CD34+ cells circulating in peripheral blood (r = 0.875). Initiating leukapheresis based on rising WBC and platelet counts rather than on a fixed day increased the mean number of CD34+ cells 115% (9.7 to 20.9 x 10(6) CD34+ cells/kg; P = 0.010) for the total of all leukaphereses and 59% for the total of all CD34-selected products (5.1 to 8.1 x 10(6) CD34+ cells/kg; P = 0.011). Although the yield and purity of the CD34-selected product were not significantly affected (P > or = 0.071), the percentage of patients with concentrations of CD34+ cells in the initial leukapheresis of > 1% increased from 47% to 70% (P = 0.004). The mean purity of the selected product was related to the starting percentage: 48.9% if < 1% and 81.5% if > or = 1% (P < 0.001). Collection of stem cells based on rising WBC and platelet counts significantly increased the number of CD34+ cells in leukaphereses and CD34-selected products in comparison with collection on a fixed day.
Bone marrow from 65 patients with aplastic anaemia (AA) was tested for stroma growth in short term cultures (2 weeks) and for colony formation by haemopoietic precursor cells during the course of their disease. In 18 untreated patients, mean stroma growth was 30% of normal and colony formation was virtually absent. After treatment with immunosuppression (IS), as estimated from 90 examinations in 54 patients, stroma growth was approximately 50% and colony growth approximately 10% of normal. Growth impairment of stroma and haemopoietic precursors persisted for 10 and more years after IS. Results of 2‐week stroma cultures were compared with results of long term bone marrow cultures in 10 AA patients and 4 controls. At 2 weeks, growth of aplastic marrow was delayed compared to normal, but this difference became less evident with prolonged incubation time. In vitro growth abnormalities were compared with the clinical evolution after IS. The development of late haematological complications (paroxysmal nocturnal haemoglobinuria (PNH)) and myelodysplastic syndrome (MDS), did not correlate with the degree of stroma growth impairment. However, relapse of aplasia was associated with poor stroma growth: 8/29 patients with stroma confluence of ≤30% during haematological remission versus 1/25 with stroma confluence of >30% relapsed. We conclude that (i) the haematopoietic microenvironment is frequently coinvolved in the disease process of AA, (ii) a defect is detected in short term rather than in long term stroma cultures and, (iii) relapse is more frequent in patients with poor stroma growth.
In vitro expression of stem‐cell factor (SCF) by bone marrow (BM) cells of 30 patients with aplastic anemia (AA) has been analyzed at the mRNA and protein levels. While no deficiencies were found in SCF mRNA expression, low levels of soluble SCF protein were measured in poorly growing AA stroma cultures. The SCF protein concentration in the supernatant and the confluence of AA stroma growth were found to correlate (R = 0.70). Defective proliferation was observed in the majority (20/30) of AA stroma cultures and was paralleled by poor growth of homogeneous cultures of fibroblasts from the same marrow sample. AA stroma growth was enhanced by addition of exogenous SCF in combination with interleukin‐11 (IL‐11), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF). Our results demonstrate that deficient growth of stroma cells results in decreased production of SCF. Therefore, SCF and other stroma‐derived cytokines may be of therapeutic value in AA patients with documented defects within the BM microenvironment.
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