Thawing of cryopreserved hematopoietic progenitor cells from apheresis with a new dry-warming device.

BACKGROUND: Current thawing techniques of cryopreserved progenitor cells are based on the use of a water bath. The aim of this study has been to assess the progenitor cell viability and the time of hematopoietic engraftment after transplantation of cell products thawed with a new dry-thawing device. STUDY DESIGN AND METHODS: In the preclinical phase, two cryobags from the same patient were thawed with the standard technique and with the dry system method in parallel (n=5, Protocol A and Protocol B, respectively). In the clinical phase, cryobags were thawed with the dry system and the time to hematopoietic engraftment after autologous transplantation (n=52) was compared with those of a control group of patients whose progenitor cell products were thawed with the standard technique (n=52). RESULTS: There were no statistical differences in nuclear and CD34+ cell viability, total colony-forming cells, and cloning efficiency after thawing with Protocols A and B. Days to neutrophil (>0.5×10(9) and >1×10(9) /L) and platelet engraftment (>20×10(9) and >50×10(9) /L) were not different between patients transplanted with products thawed with Protocols A and B. CONCLUSION: Progenitor cell viability and function are preserved with this dry-thawing system. The time to hematopoietic engraftment of patients after transplantation is comparable to those infused with progenitor cells thawed with the water bath technique. Thawing cell products without the use of water and in a dry environment might favor the use of this dry method.

Cellular transplants in amyotrophic lateral sclerosis patients: an observational study.

BACKGROUND AIMS: Cytotherapy is a promising option for neurodegenerative disease treatment. Because of the fatal prognosis and imperative need for effective treatment, amyotrophic lateral sclerosis (ALS) patients request this therapy before its effectiveness has been verified. The increase in clinics offering cytotherapies but providing little scientific information has prompted considerable medical tourism. We present an observational study of Spanish ALS patients receiving cytotherapy, analyzing the experiences arising from the treatment (TX) and considering two progression markers, FVC and ALSFRS-R. METHODS: Twelve ALS patients with a mean age of 48.6 years (SD 12.8) received cytotherapy 26.9 months (SD 15.8) after clinical onset. ALSFRS-R and FVC at TX were 32.3 (SD 6.8) and 63.4% (SD 15.3), respectively. TX involved transplants of olfactory ensheathing cells in three patients, and autologous mesenchymal stromal cells in the remainder. RESULTS: One patient died 33 months post-TX after surviving for 49 months. Five required mechanical non-invasive home ventilation 7.4 months post-TX. Two required invasive ventilation 13 months post-TX. Five patients needed gastrostomy feeding 23.3 months post-TX. Survival between clinical onset and the study end date was 50 months (SD 17.2). No significant adverse events or changes in the decline of FVC and ALSFRS-R compared with the disease's natural history were observed. CONCLUSIONS: Our observations suggest that these therapies do not halt the course of the disease. Cytotherapy cannot yet be considered a curative treatment for ALS.

Combined intermittent hypoxia and surface muscle electrostimulation as a method to increase peripheral blood progenitor cell concentration.

BACKGROUND: Our goal was to determine whether short-term intermittent hypoxia exposure, at a level well tolerated by healthy humans and previously shown by our group to increase EPO and erythropoiesis, could mobilize hematopoietic stem cells (HSC) and increase their presence in peripheral circulation. METHODS: Four healthy male subjects were subjected to three different protocols: one with only a hypoxic stimulus (OH), another with a hypoxic stimulus plus muscle electrostimulation (HME) and the third with only muscle electrostimulation (OME). Intermittent hypobaric hypoxia exposure consisted of only three sessions of three hours at barometric pressure 540 hPa (equivalent to an altitude of 5000 m) for three consecutive days, whereas muscular electrostimulation was performed in two separate periods of 25 min in each session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment and 24 h, 48 h, 4 days and 7 days after the last day of hypoxic exposure. RESULTS: There was a clear increase in the number of circulating CD34+ cells after combined hypobaric hypoxia and muscular electrostimulation. This response was not observed after the isolated application of the same stimuli. CONCLUSION: Our results open a new application field for hypobaric systems as a way to increase efficiency in peripheral HSC collection.

Cryopreservation of hematopoietic progenitor cells from apheresis at high cell concentrations does not impair the hematopoietic recovery after transplantation.

BACKGROUND: The high number of nuclear cells (NCs) from hematopoietic progenitor cells-apheresis (HPC-A) requires cryopreservation in large volumes or at high NC concentrations. The effect of NC concentration during cryopreservation has yet to be examined. STUDY DESIGN AND METHODS: In the experimental arm (n = 610, Protocol B), the first HPC-A sample from the patient was cryopreserved in two cryobags and subsequent collections in one cryobag, resulting in high NC concentrations (>100 x 10(6) NCs/mL) in most cases. The effect of NC concentrations at freezing in NC recovery after thawing and engraftment kinetics was analyzed and compared with a group of HPC-A cryopreserved at standard NC concentrations (n = 455, Protocol A). RESULTS: The mean (SD) NC concentration at freezing was 78 (28) x 10(6) per mL (median, 82 x 10(6)/mL; range, 12 x 10(6)-156 x 10(6)/mL) and 183 (108) x 10(6) per mL (median, 156 x 10(6)/mL; range, 16 x 10(6)-678 x 10(6)/mL), for HPC-A cryopreserved according to Protocols A and B, respectively. The NC viabilities of the test vials and HPC-A components after thawing were 88 percent versus 85 percent and 85 percent versus 82 percent, and the cloning efficiency was 49 percent versus 33 percent for Protocols A and B, respectively (p < 0.001). Significant differences were not observed in the recovery of NCs. Days to neutrophil and platelet engraftment were not different between patients transplanted in the standard- (n = 143) or high-cell-concentration group (n = 238). CONCLUSION: The cryopreservation of HPC-A at higher than standard NC concentrations has no adverse impact on hematopoietic reconstitution after transplantation.

Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing.

BACKGROUND: The direct transfusion of thawed hematopoietic progenitor cells (HPCs) is associated to transfusion-related side effects that are thought to be dose-dependent on the infused dimethyl sulfoxide (DMSO). Both the effectiveness of a fully automated cell processing device to washing out DMSO and the effects of DMSO elimination over the recovered cells were evaluated. STUDY DESIGN AND METHODS: Twenty cryopre-served peripheral blood HPC bags (HPC apheresis [HPC-A]) were thawed and processed for washing with an automated cell-processing device. Viability, colony-forming units (CFUs), and absolute count of recovered cells were evaluated by flow cytometry immediately after washing as well as at different times after washing and compared with a sample taken just after thawing (control) but maintained at 4 degrees C. DMSO content was measured by high-performance liquid chromatography and the osmolarity with an osmometer. RESULTS: The median recovery of viable total nucleated cells, viable CD34+ cells, and CFU colonies was 89 (range, 74-115), 103 (range, 62-126), and 91 percent (range, 46%-196%), respectively, in the washing group. Recovery of viable CD3+ cells was 97 percent (range, 42%-131%) and CD14+ cells was 82 percent (54%-119%). The percentages of DMSO elimination and osmolarity reduction were 98 (range, 96-99) and 90 percent (range 86%-95%), respectively. Moreover, elimination of the cryoprotectant improved CFU count, viability, and cell recoveries along the time when compared with the control group. CONCLUSION: Washing out DMSO in thawed HPC-A by use of this approach is safe and efficient in terms of recovery and viability of nucleated and progenitor cells. Additionally, the removal degree of DMSO is very high and therefore might ameliorate the transfusion-related side effects.

Mixed chimerism is frequent after allogeneic peripheral blood stem cell transplantation with positive CD34 selection, and is not reverted by low doses of donor T-cells add-back.

UNLABELLED: The incidence of full donor chimerism (full DC) after CD34+ -selected peripheral blood stem cell transplantation (CD34+ -PBSCT) is controversial. Whereas the initial reports suggested a high incidence of full DC (hypothetically because of the high number of CD34+ cells infused) more recent works describe a high incidence of mixed lymphoid chimerism. There are no data concerning the ability of low-dose donor T-lymphocyte add-back on conversion to full DC. METHODS: We prospectively monitored the chimerism status of 25 patients undergoing CD34+ -PBSCT and the effect on chimerism of delayed low doses of donor T-cell add-back (TCAB). One, two or three doses of TCAB were administered on days +28 (2 x 10(5) CD3+/kg), +60 (2 x 10(5) CD3+/kg) and +90 (2 x 10(6) CD3+/kg), respectively, when on cyclosporine A prophylaxis. RESULTS: Incidence of full DC on day +20 was 56%. However, all but two patients progressed to MC. Fifteen patients were scheduled to TCAB. Six patients with initial MC did not convert to full DC after TCAB. Moreover, seven patients with full DC status progressed to mixed chimerism. CONCLUSIONS: Our results indicate that low doses of TCAB administered under cyclosporine A prophylaxis have no effect on the eradication of the recipient cells. We believe that a high dose of CD34+ cells in the grafts of CD34+ -PBSCT is not enough to achieve stable full DC unless a minimum number of CD3+ cells are infused, or more intensified transplant conditioning regimens are employed.

Strategies to reduce transplant-related mortality after allogeneic stem cell transplantation in elderly patients: Comparison of reduced-intensity conditioning and unmanipulated peripheral blood stem cells vs a myeloablative regimen and CD34+ cell selection.

OBJECTIVES: The aim of this study was to compare two approaches used to reduce transplant-related mortality (TRM) after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) in elderly patients. PATIENTS AND METHODS: Data from 50 patients, 45 years of age or older, consecutively treated with an HLA-identical sibling allo-PBSCT at the Hospital de Sant Pau were analyzed. We have compared the outcome of patients treated with conventional myeloablative regimens and CD34(+)-selected cells (CD34(+) group; n=23) with those receiving reduced-intensity conditioning regimens, consisting of fludarabine (150 mg/m(2)) plus an alkylating agent, followed by unmanipulated grafts (RIC group; n=27). Patient characteristics were well balanced between the two groups, although patients in the RIC group were slightly older. RESULTS: The incidence of acute graft-vs-host disease (GVHD) was similar in both groups. The 1-year cumulative incidence of extensive chronic GVHD was 38% in the RIC group and 17% in the CD34(+) group (p=0.2). After a median follow-up of 28 months, there were no differences in the relapse rate. Patients in the RIC group had a lower TRM, with a cumulative incidence of 7% vs 30% at 6 months and 15% vs 39% at 1 year (p=0.05). The Kaplan-Meier estimates of PFS at 2 years was 67% in the RIC group and 43% in the CD34(+) group (p=0.09) and the OS was 69% vs 43% (p=0.05), respectively. CONCLUSION: CD34(+) cell selection reduced the risk of extensive cGVHD but was associated with a higher TRM. Although the number of patients is limited, our study suggests that this approach should be restricted to relatively young patients, as better outcomes can be achieved in elderly patients using RIC strategies.

CD34+-enriched-CD19+-depleted autologous peripheral blood stem cell transplantation for chronic lymphoproliferative disorders: high purging efficiency but increased risk of severe infections.

OBJECTIVE: The main objective of this work was to decrease the incidence of relapse after autologous stem cell transplantation with a "double purging" procedure. METHODS: We used a "positive" (CD34) and "negative" (CD19) double selection method to improve the efficacy of "single purging" of hematopoietic harvests in poor-prognosis lymphoproliferative disorders. All patients included in the study had a positive molecular marker of their disease. Minimal residual disease (MRD) was studied by flow cytometry and PCR techniques during the purging procedure and after transplantation. RESULTS: Twenty-six patients fulfilled entry criteria. Median age of patients was 50 years (range: 33-66); 17 were male and 9 female. Thirteen (50%) of the patients mobilized an adequate number of CD34+ cells (>or=3 x 10(6)/kg) to proceed with the double-selection protocol. Twelve of the 13 harvests became PCR negative after purging. Ten patients were grafted with the selected products and all but one engrafted without delay. After a median follow-up of 30 months, 2 of 10 patients suffered a molecular relapse at 7 and 19 months respectively. The earlier relapse was observed in the patient who received a MRD+ product. Only one patient experienced a clinical relapse. Three patients died due to obliterans bronchiolitis, pneumococcal sepsis, and septic shock of unknown origin, respectively, and three others presented life-threatening infections. CONCLUSION: Therefore, CD34+/CD19+ positive/negative selection is an effective purging approach in patients with chronic lymphoproliferative disorders. This favorable effect is, however, counterbalanced by the high frequency of life-threatening infections.