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Introduction: In order to propose risk-adapted mobilization algorithms, several authors have tried to look for predictive factors of the CD34 + yield in healthy pediatric donors. Donor recipient body weight ratio (D/R ratio) was identified as one of the main variables related with the success to achieve the target cell dose for transplantation. According to this variable we modified the mobilization schedule., Material and Methods: We report the results of 46 mobilizations and apheresis procedures performed in our center with unfavorable D/R ratio. Mobilization was attempted by the standard regime of G-CSF (10 mcg/kg/24 hours) in 28 cases (60.9%), with high dose G-CSF (10 mcg/kg/12 hours) in 9 cases (19.6%), and with plerixafor and G-CSF single dose regime in 9 cases (19.6%)., Results: CD34 + cell quantification before apheresis is closely related to CD34 + yield, being the only factor related to collected CD34 + cells (beta .71; P < .0001). The mobilization efficiency was higher in plerixafor group compared to the other two schedules (P < .0001). By using plerixafor for mobilization, we achieved the target CD34 + cell dose of ≥2 × 10 6 /kg per recipient body weight in all cases with unfavorable D/R ratio. It was observed that 17.4% of cases that not reached the established target cell dose were located in the standard or high-dose mobilization regimes. This difference is even greater for optimal collections (≥5 × 10 6 /kg), since of the 54.3% cases that did not reach this goal none was mobilized by plerixafor., Conclusion: Tailoring the mobilization regime we can reach the target cell dose, even in those cases with the worst D/R ratio., (© 2020 Wiley Periodicals LLC.)

The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here, we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFκB pathway (the NFκB inducing kinase, NIK, and the downstream molecule NFκB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy, and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation); however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild-type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, that is, either NFκB2 or c-Rel, only NFκB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFκB pathway for the recovery of normal levels of hematopoiesis after stress., (© 2015 AlphaMed Press.)

Plerixafor has been recently approved by the European Medicines Agency for adult patients who have failed other mobilization strategies. Experience in children, however, is extremely limited. We describe the experience of the use of this drug in 8 children under a compassionate-use program in 3 Italian and 2 Spanish centers. Plerixafor was generally well tolerated; only 2 of 8 children reported adverse effects, and these were mild in intensity. Peripheral blood progenitor cell priming was improved with plerixafor in 6 of 8 patients. In the remaining 2 patients, the target CD34+ cell count was below the target of 2 × 10(6) cells/kg, although in these patients cell counts before collection were good enough for leukapheresis. Plerixafor, therefore seems to be safe and effective for peripheral blood progenitor cell mobilization in children. Adverse events were comparable with those described with filgrastim alone.

Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

The use of children as haematopoietic stem cell donors represents an ethical dilemma. For some investigators, this is even more questionable if the children underwent peripheral blood progenitor cell (PBPC) collection. Due to these controversies, information related to adverse events (AE) in paediatric donors and variables related to the collection is scanty. We analysed 152 PBPC collections, and compared the complications and results between young children, older children and adults. The pattern of AE was shown to vary according to the age of the donor. Older paediatric donors and adults had a higher incidence of complaints related to PBPC priming (54.3% vs. 79.7%, respectively) than the youngest children (12%). On the other hand, these donors had a lower incidence of AE during PBPC collection (19.6% older children, 37.3% adults) mainly related to hypocalcaemia, than the youngest donors, who suffered mainly cardiovascular complications due to hypovolaemia (51.7%). The only variables related to collected cell dose were total blood volume processed per donor body weight, and CD34(+) cell count before apheresis. The donor/recipient body weight ratio predicted the outcome of collection in a single large volume leukapheresis. Donors with body weight ratio >or=0.75 had 4.69 times higher likelihood to reach the minimum target cell dose.

Background: Peripheral blood progenitor cell (PBPC) collection has become the main source of hematopoietic cells for high-dose chemotherapy with stem cell rescue and, in some protocols, for allogeneic hematopoietic transplantation. This procedure is complicated in the smallest children because of difficulties related to their weight, and there is little published experience. We have conducted a prospective study to analyze the incidence of adverse events during PBPC collection in the smallest children (< or = 10 kg)., Methods: From January 2000 to November 2005, 257 leukapheresis were performed in our unit, and 13 of them (5%) in 12 children weighing up to 10 kg (median 9 kg, range 5.8-10.9 kg)., Results: Most cases had hypovolemic signs during the procedure (usually tachycardia); six cases had hypotension, five of them with pallor and diaphoresis, and, of those, two also had nausea. In all these cases infusion of saline or plasma volume expanders resolved the clinical findings. In two cases the nausea related to hypocalcemia was resolved after calcium gluconate infusion. Changes in platelet counts were also remarkable, with a median platelet loss of 52%., Discussion: Leukapheresis with continuous-flow cell separators has frequent complications related to volume shift in the smallest children. These adverse events are mild and easily resolved with standard measures for hypovolemia, as plasma expander or normal saline infusions. However, we recommend that the procedure should only be performed by teams with extensive experience in the field.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by genetic instability of cells lacking a functional FA/BRCA pathway. Previous studies have shown that in vitro stimulation of bone marrow cells (BMCs) from FA mice promotes apoptosis, reduces the reconstitution ability of the stem cells, and induces myelodysplasia and myeloid leukemia upon reinfusion of the cells. This suggests the convenience of adapting standard protocols of gene therapy to FA. Here we show that the reserve of BM progenitors in FA patients is generally below 20% of normal values. Because this reduced reserve could activate the cycling of BM progenitors, we developed a simplified protocol to transduce BMCs from FA patients with gammaretroviral vectors. We demonstrate that a short in vitro manipulation (12-24 hr) of fresh mononuclear BMCs is sufficient to transduce 42% of hematopoietic progenitors from FA-A patients, in the absence of in vitro prestimulation. When FANCA-expressing vectors were used, this simple procedure reversed the phenotype of the BM progenitors from these patients. We propose that our approach will be more efficient and safer compared with standard gene therapy protocols for FA.

Peripheral blood progenitor cells (PBPC) are increasingly used as a source of stem cells for either autologous or allogeneic hematopoietic transplantation in children. Although technically similar to adult procedures, apheresis in small children worries some operators and physicians that have little experience in managing pediatric patients or donors. However several published series have showed that these procedures, when performed by experienced teams are safe and can obtain enough amount of PBPC for either autologous or allogeneic hematopoietic transplantation. Some technical aspects must be considered in pediatric apheresis due to the size of the patient/donor. Factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. Other important issue to be considered is the emotional stress for them and their relatives. Leukaphereses in children may be performed with any of the reported continuous blood cell separator without sedation. Adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. Anyway, taking all these complications together, they are not higher than those adverse events or toxicities reported after general anesthesia and bone marrow harvesting.

Large volume leukapheresis (LVL) has been proposed as a simplified single-apheresis approach to collect the target number of CD34(+) cells. We retrospectively analyzed results of LVL in cytokine-mobilized patients weighing less than 20 kg to evaluate adverse events and variables affecting the yield. The only major adverse event recorded was transient and reversible systolic hypotension (three episodes). All the other adverse events were mild and did not require treatment. In multivariate analysis leukocyte count (P=0.001) and younger age (P=0.009) affected the CD34(+) cell number in the peripheral blood before apheresis. The number of CD34(+) cells before the apheresis was the only variable affecting CD34(+) cell yield in multivariate analysis (P=0.0001). In all, 77% of patients achieved the target CD34(+) cell dose of 2 x 10(6)/kg in their first apheresis. Recruitment was seen in 72% of the procedures, and this was related to the total blood volume processed (P=0.0005).

Peripheral blood progenitor cells (PBPC) are substituting bone marrow as a source of stem cells for either autologous or allogeneic hematopoietic transplantation. Several papers have been published on the experience of various groups in their mobilization and transplantation in children. Some technical problems have derived from the size of the patient or donor in the pediatric setting. Thereby, there is some concern regarding leukapheresis in very small children (weighing less than 15-20 kg). This paper summarizes our own data and that of other groups for the mobilization and collection of PBPC in the smallest children. Data from the literature show that mobilization with cytokines alone or in combination with chemotherapy is well tolerated by these patients. Pediatric donors may be used for allogeneic transplantation with no higher incidence of complications. PBPC collection even in the smallest children is a safe and efficient procedure when performed by experienced apheresis teams.

Background and Objectives: We analyzed the relationship between long-term hematopoietic recovery and the number of CD34+ cells infused in order to determine the optimal dose of CD34+ cells for rapid and stable engraftment., Patients and Methods: Between November 1993 and December 1998, 96 consecutive autologous transplantations were performed in 92 pediatric patients with different malignancies. Peripheral blood progenitor cells (PBPC) were mobilized by G-CSF alone (12 microg/kg/day s.c., Neupogen((R)); Amgen, Thousand Oaks, Calif., USA) and collected using a Cobe Spectra blood cell separator (Cobe, Denver, Colo., USA) through a central venous catheter with double lumen. The CD34+ cell contents of apheresis products were assessed by means of flow-cytometric analysis using an Epics Elite flow cytometer (Coulter, USA)., Results: The median number of CD34+ cells infused was 3.2 x 10(6)/kg (range 0.17-44.4). The median times for short-term engraftment (neutrophil count >0.5 x 10(9)/l and platelet count >20 x 10(9)/l) was 9 (range: 7-16) and 13 days (range: 7-91), respectively. The median times for long-term engraftment (platelet count >50 x 10(9)/l and >100 x 10(9)/l) was 21 (range: 10-249) and 45 days (range: 12-288). When the infused CD34+ cell dose was >/=5 x 10(6)/kg (median 7.99, range 5.01-44.4), there was a statistically significant increase in the rate of short- and long-term hematopoietic recovery compared to patients transplanted with a lower number of CD34+ cells (p < 0.0001). The earlier recovery in the high CD34+ cell group resulted in less transfusional support, fewer days on intravenous antibiotics and shorter hospitalization., Conclusions: This study confirms that G-CSF-mobilized PBPC provide rapid short- and long-term hematopoietic engraftment in pediatric patients undergoing autologous transplantation if a CD34+ cell dose >/=5.0 x 10(6)/kg is infused. As this PBPC dose seems to have clinical and potentially economic implications, it should be considered the optimal dose for apheresis., (Copyright 2000 S. Karger AG, Basel)

Background and Objective: In children it is very important to optimize PBPC harvesting and to reduce the number of leukaphereses per patient. The value of pre-apheresis peripheral blood CD34+ cell concentration as a predictor of PBPC yield was studied in 23 pediatric patients with hematologic and non-hematologic malignancies in order to optimize duration of PBPC collection., Design and Methods: The patients underwent 25 stem-cell mobilization episodes with G-CSF alone and 40 large-volume leukapheresis procedures. Peripheral blood and harvested CD34+ cell concentrations were analyzed by means of flow cytometry., Results: Using linear regression analysis, a highly significant correlation was found between the peripheral blood CD34+ cell count and the CD34+ cells/kg patient body weight collected on the apheresis day (r = 0.826, p = 0.0001). The results indicate that at least 1 x 10(6)/kg CD34+ cells can be harvested during one leukapheresis procedure in all patients if the pre-apheresis blood CD34+ cell count is > or = 30/microL and a CD34+ cell target of > or = 5 x 10(6)/kg is achieved in at least 80% of patients if this value is > or = 50 CD34+ cells/microL processing a median blood volume of 438.7 mL/kg (range, 207-560) over a median time of 232.5 minutes (range, 182-376)., Interpretation and Conclusions: Our results suggest that the number of CD34+ cells harvested in a single large-volume leukapheresis can be predicted from the measurement of peripheral blood CD34+ cell concentration on the collection day.

The main objective of the present study was to determine the role of CD34+ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G-CSF were analysed. Using dual-colour flow cytometry, different subpopulations of CD34+ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34+CD38- cells and CD34+CD38+ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34+ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 X 10(6) CD34+CD38- cells for neutrophil engraftment, and 2.0x10(6) CD34+CD38+ cells for platelet recovery. It is suggested that the analysis of CD34+ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34+ cells are collected.

Large-volume leukapheresis (LVL), defined as the processing of at least three blood volumes in a single session for peripheral blood progenitor cell (PBPC) collection, was performed in 32 small children weighing < or = 25 kg, aged 10 months to 8 years, with a variety of malignancies. Harvesting of PBPC was started after 4 days of cytokine (G-CSF, 12 micrograms/kg s.c.) alone. Procedures were performed using a continuous flow blood cell separator (COBE Spectra). The automated program of lymphocytapheresis was modified to achieve a collection rate of 0.9 ml/min. The extracorporeal line was primed with a unit of a packed red blood cells before the procedure. Acid citrate dextrose (ACD) was used as anticoagulant with an ACD inlet ratio of 1:14 and an ACD infusion rate of 1.1 ml/min/L of total blood volume. The inlet flow ranged between 6 and 35 ml/min (median 20 ml/min). A total of 37 apheresis procedures were performed (median 1, range 1-3). In 84% of patients, a single apheresis yields the minimum number of PBPC cells required for transplantation. No consistent side effects were observed, and LVL was well tolerated by children. A median of 7.7 x 10(8) kg MNC, 5.4 x 10(6)/kg CD34+, and 6.2 x 10(4)/kg CFU-GM per apheresis were harvested. Patients with neuroblastoma had a significantly lower yield than other patients. To date, 27 patients have been transplanted after myeloablative treatment, and rapid and sustained engraftment was achieved in all cases. The number of CD34+ cells infused was highly correlated with engraftment kinetics. LVL can be safely and easily performed in small children, allowing adequate PBPC collection for transplantation with rapid hematologic recovery.

The aim of this study was to analyze factors affecting mobilization and engraftment in 40 children undergoing autologous peripheral blood progenitor cell transplantation for different malignancies: 19 patients with haematological malignancies and 21 patients with solid tumors. Patients received 4-5 days of rhG-CSF (12 micrograms/kg/day) subcutaneously. Apheresis was performed by continuous flow blood cell separation beginning on the fifth day of rhG-CSF. For patients weighing < or = 25 kg, the extracorporeal line was primed with irradiated red blood cells. After myeloablative conditioning regimens, patients were grafted with 7.21 +/- 7.8 x 10(6)/kg CD34+ cells. Days to achieve an absolute neutrophil count > 0.5 x 10(9)/1 and a platelet count > 20 x 10(9)/1 without platelet support were 9.50 +/- 1.2 (range 7-13) and 18.1 +/- 8.3 (range 9-37), respectively. The number of CD34+ cells infused was highly correlated with engraftment kinetics (P = 0.0001). The patient's body weight and the number of previous chemotherapy courses had a negative influence on CD34+ cells collected.

Large volume leukapheresis (LVL) defined as the processing of greater than three volumes of blood in a single session for peripheral blood stem cell (PBSC) collection was performed in 27 children, aged from 1 to 15 years, with various malignancies. Harvesting of PBSC was started after 4 days of cytokine (G-CSF 12 micrograms/kg s.c.) alone. With the exception of two cases the rest (92.5%) needed only a single apheresis to yield the minimum number of cells required for transplantation. No consistent side-effects were observed and the LVL were well tolerated by children. An average of 7.6 x 10(8) MNC/kg, 6.1 x 10(6)/kg CD34+ and 2.1 x 10(4)/kg CFU-GM were harvested. To date, 19 patients have been transplanted after myeloablative treatment and sustained engraftment was achieved in all cases. LVL can be safely and easily performed in children allowing adequate PBSC collection for transplantation with prompt hematological engraftment.

Donor killer cell immunoglobulin-like receptor (KIR)-ligand incompatibility is associated with decreased relapse incidence (RI) and improved leukemia-free survival (LFS) after haploidentical and HLA-mismatched unrelated hematopoietic stem cell transplantation. We assessed outcomes of 218 patients with acute myeloid leukemia (AML n=94) or acute lymphoblastic leukemia (n=124) in complete remission (CR) who had received a single-unit unrelated cord blood transplant (UCBT) from a KIR-ligand-compatible or -incompatible donor. Grafts were HLA-A, -B or -DRB1 matched (n=21) or mismatched (n=197). Patients and donors were categorized according to their degree of KIR-ligand compatibility in the graft-versus-host direction by determining whether or not they expressed HLA-C group 1 or 2, HLA-Bw4 or HLA-A3/-A11. Both HLA-C/-B KIR-ligand- and HLA-A-A3/-A11 KIR-ligand-incompatible UCBT showed a trend to improved LFS (P=0.09 and P=0.13, respectively). Sixty-nine donor-patient pairs were HLA-A, -B or -C KIR-ligand incompatible and 149 compatible. KIR-ligand-incompatible UCBT showed improved LFS (hazards ratio=2.05, P=0.0016) and overall survival (OS) (hazards ratio=2.0, P=0.004) and decreased RI (hazards ratio=0.53, P=0.05). These results were more evident for AML transplant recipients (2-year LFS and RI with or without KIR-ligand incompatibility 73 versus 38% (P=0.012), and 5 versus 36% (P=0.005), respectively). UCBT for acute leukemia in CR from KIR-ligand-incompatible donors is associated with decreased RI and improved LFS and OS. [ABSTRACT FROM AUTHOR]

Summary:Several genetic diseases, generally considered as congenital diseases, are characterized by bone marrow failure during early childhood. Hematopoietic stem cell transplantation is the only curative treatment for syndromes involving bone marrow failure and thalassemia. In this slate-of-the-art review, we wish to focus on the results of hematopoietic transplantation in treating some of these diseases, with a special emphasis on congenital bone marrow failure and thalassemia. The results of this procedure have improved over the previous years, mainly when performed by experienced teams. New conditioning regimes based on fludarabine and the use of HLA-identical donors have been related with better survivals. In the previous years, donors other than HLA-identical siblings have been increasingly used in patients not responding to conventional measures, but this approach needs to be evaluated in larger studies.Bone Marrow Transplantation (2005) 35, S17-S21. doi:10.1038/sj.bmt.1704838 [ABSTRACT FROM AUTHOR]