A new system for quality control in hematopoietic progenitor cell units before reinfusion in autologous transplant.

BACKGROUND: In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis (HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors. STUDY DESIGN AND METHODS: This study was designed to assess the potential of an automatic cell counting system (NucleoCounter NC-3000, ChemoMetec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with NucleoCounter NC-3000, by using two protocols: "vitality assay" and "mitochondrial potential assay." RESULTS: The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) assay showed a negative correlation (r=-0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 10(6) /L viable total nucleated cells. We observed a significant difference (p<0.0001) comparing the median number of colonies (166.70; SD, ± 136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ± 59.76) obtained with a value of JC-1 more than 30%. CONCLUSION: The evaluation of cell functionality by the use of the NucleoCounter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory.

Optimization of the immunomagnetic selection in microcythemic donors enrolled for haploidentical transplantation.

BACKGROUND: Immunomagnetic cell selection (ICS) cells is increasingly used in allogeneic hematopoietic transplantation in order to reduce the T cells quantity. The aim of this study was to evaluate an protocol based on Ficoll method before ICS. STUDY DESIGN AND METHODS: The automated procedure was compared with the standard method. In the group 1 the cell processing involves the extraction of the buffy-coat by Ficoll before incubation with antibodies. This procedure was performed with the Sepax S-100 device. The efficacy of this automated procedure was compared with the group 2. In this group, the cell washing and the incubation were performed through the standard method. The CD34+ cells collected by apheresis (HPC-A) were selected with ICS. RESULTS: The results obtained after Ficoll procedure, showed a total nucleated cells (TNCs) and CD34+ cells recovery of 85.73% (75.90-90.63; SD 4.25) and 79.31% (51.77-112.31; SD 18.40), respectively. The TNC and CD34+ cells recovery after the pre-incubation washing performed through the standard method, was 75.54% (38.36-97.76; SD 22.5) and 61.51% (30.87-81.79; SD 19.3), respectively. The CD34+ cells recovery after ICS was 79% (51.77-100; SD 18.40) and 44% (15.57-88.24; SD 25.91) in the group 1 and the group 2, respectively. This difference was statistically significant (p=0.001). CONCLUSION: The efficacy of the ICS which resulted to be higher in the group 1 compared to the group 2. Overall, our data suggest that the Ficoll procedure before incubation is suitable for the clinical routine in the ICS for haploidentical transplantation in patients affected by thalassemia.

Cell processing for haplo-identical hematopoietic stem cell transplantation: automated washing and immunomagnetic-positive selection.

BACKGROUND AIMS: Immunomagnetic cell selection (ICS) of CD34(+) cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection. METHODS: The automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The efficacy of the automated procedure was compared with the control group, where washing were performed using a standard method. RESULTS: The results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34(+) cell recovery of 84.87% (71.80-105, SD 8.62; range, standard deviation) and 83.45% (47-109, SD 16.12), respectively. The NC and CD34(+) cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36-97.76, SD 22.5) and 61.51% (30.87-81.79, SD 19.3), respectively. The CD34(+) cell recovery after ICS was 51.27% (13.77-98.82, SD 24.97) and 48.89% (15.57-88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4-96.10, SD 13.07) and in group 2 84.97% (58.1-97.8, SD 15.58). CONCLUSIONS: The efficacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines.

Evaluation of cell death after treatment with extracorporeal photopheresis.

The aim of our study is to assess the mortality of leukocytes during extracorporeal photopheresis. Sixty-three photopheresis performed on 13 patients affected by chronic GvHD were evaluated. Samples were analyzed using a FACSCalibur flow cytometer. Apoptosis and necrosis of limphomononuclear cells dramatically increased after the apheretic procedure. We found a further increase of apoptotic and necrotic limphomononuclear cells after treatment with 8-MOP and UVA (p≤0.05). Our data suggested that the immunomodulatory effects of extracorporeal photopheresis, triggered by circulating apoptotic or necrotic cells, could play an important role in the treatment of GvHD with this procedure.

Transplantation in the onco-hematology field: focus on the manipulation of αß and γδ T cells.

γ/δ T cells represent a subset of T cells expressing a T cell receptor (TCR) variant composed of gamma and delta chains. The γ/δ TCR is expressed by 2-10% of all T cells in human peripheral blood, whereas the majority of T cells express α/ß TCRs. γ/δ T cells display a range of innate effector functions including rapid secretion of chemokines and cytokines, as well as target cell lysis. Recent interest has focused on the function of γ/δ T lymphocytes in allogeneic transplantation in the onco-hematology field. Several studies, in vitro and in vivo, suggest that γ/δ T lymphocytes are potential beneficial effector cells in the context of hematopoietic stem cell transplantation (HSCT). In addition, in this review, we discuss the depletion of α/ß T lymphocytes in the graft for allogeneic transplantation. In fact, an efficient TCR α/ß cell depletion potentially reduces the risk of GvHD. Furthermore, TCR α/ß T cell depletion, especially with immunomagnetic negative selection, retains other potential beneficial effector cells in the graft, such as γ/δ T cells, NK cells, and stem cells. These "facilitating" cells might facilitate engraftment, exert GvL effects, and reduce the risk for infections.

Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system.

BACKGROUND: Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space. STUDY DESIGN AND METHODS: The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated. RESULTS: The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing. DISCUSSION: Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.

T(reg) cells: collection, processing, storage and clinical use.

T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of T(reg) cells designated naturally occurring and induced. Interest in T(reg) cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding T(reg) cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127(low) FOXP3+ regulatory T cells.

Pre-transplant manipulation processing of umbilical cord blood units: Efficacy of Rubinstein's thawing technique used in 40 transplantation procedures.

BACKGROUND: Umbilical cord blood (UCB) is a valid alternative to be used in transplanted patients. Limitations of the use of stem cells depends on the small number of cells available; this is the reason why UCB can be used only in very low-weight patients. In this study we have evaluated the efficacy of cellular manipulation before transplant and in particular, before thawing the units through the Rubinstein method. METHODS: We have evaluated the results obtained after thawing 40 UCB to be used for as many patients affected by several pathologies (21 ALL, 6 AML, 3 MDS, 2 LNH, 2 histiocytosis, 2 ß-thalassemia, 1 Chédiak-Higashi syndrome, 1 Fanconi anemia, 1 Wiskott-Aldrich syndrome and 1 Omenn syndrome). RESULTS: After thawing, nucleated cells (NC) mean recovery was 76.81% (SD±15.41). The quantity of NC obtained was 124.29×107 (SD±43.18) and in only 5 cases the number of NC after the procedure was lower than the requested graft dose. Among the last ones, in two cases only we did not achieve the target after manipulation. The post-manipulation cellular viability was 83.48% (SD±10.6). For all the units shipment complied with all the necessary procedures; in fact the temperature never rose above -120°C. CONCLUSION: In our study we highlighted the efficacy of UCB thawing technique, with the same method defined in 1995 at the New York Blood Centre that guarantees an excellent NC recovery and maintains a high level of cell viability.

Immunohematologic reconstitution in pediatric patients after T cell-depleted HLA-haploidentical stem cell transplantation for thalassemia.

To analyze immunohematologic reconstitution, particularly of natural killer (NK) cells, we evaluated 13 ß-thalassemia patients after 20 and 60 days and 1 year posttransplantation with T cell-depleted HLA-haploidentical stem cells. We assessed lymphocyte and bone marrow (BM) progenitor cell phenotype and differentiation capacity, spontaneous BM cytokine production, stromal cells, and stromal cell interleukin (IL)-7 production. A reduced clonogenic capability manifested at day +20. Patients had significantly lower CD4(+) T cells versus controls, mainly in the CD45RA(+)CD62L(+) subset. NKs were among the first lymphocytes to repopulate the peripheral blood. At day +60, an increase in primitive BM progenitor cells paralleled small increases in CD4(+), naïve CD4(+), and thymic naïve Th cells. A significant increase in CD4(+) and CD8(+) markers paralleled an increase in CD3⁻CD16(+) NKs, especially with full engraftment. In patients with stable mixed chimerism we observed very low levels of CD3(+) donor chimerism early after transplant that increased over time, but a stable population of high donor NK cells, suggesting a role of these cells on donor engraftment. Stromal cells secreted less IL-7 and displayed "macrophage-like" morphology. Patients initially manifested impaired stem/progenitor cell growth and differentiation capacity in parallel with altered T cell homeostasis and a reduced T cell naïve compartment. We hypothesize that T cell compartment damage partly arises from altered new T cell production from the hematopoietic stem/progenitor cells under stromal cytokine influence. NNK subset analysis might be useful for determining transplant outcome.

Positive immunomagnetic CD34(+) cell selection in haplo-identical transplants in beta-thalassemia patients: removal of platelets using an automated system.

BACKGROUND AIMS: Immunomagnetic CD34(+) cell selection (ICS) is utilized in autologous and allogeneic transplants. In the first case it is used to reduce the neoplastic contamination of concentrates, while in the second case it is needed to carry out a T-depletion of cell concentrates in order to reduce the incidence of graft-versus-host disease (GvHD) in patients who have undergone haplo-identical transplants. METHODS: The efficacy of CliniMACS technology, after reduction of platelet contamination, incubation of monoclonal antibodies (MAb) and successive washings of concentrates, performed in 16 ICS using the standard method without reducing platelet content, was compared with the use of the automated system CytoMate, which was carried out in 46 ICS. RESULTS: In the group of ICS carried out after automatic manipulation, a significant statistical difference in purity was noted (91.39% versus 83.57, P = 0.017) compared with the group of ICS carried out with the standard procedure. The same significant difference was noted in relation to the remaining percentages of CD3(+) and CD19(+) cells (2.31% versus 5.68%, P = 0.012, and 1.58% versus 2.71%, P = 0.014, respectively). Recovery of CD34+ cells overlapped in the two groups (70.49% versus 68.39%, P = 0.774). CONCLUSIONS: Immunomagnetic selection carried out using the automated procedure was more efficient, producing a purer sample, more efficient T-depletion and optimal reduction of B cells, without influencing cell recovery. Furthermore, conforming to good manufacturing practice (GMP) guidelines, the entire procedure with CytoMate took place in a contamination-controlled environment.

Purified T-depleted, CD34+ peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia.

Fetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen-identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day -59 to -11; 30 mg/m(2) fludarabine from day -17 to -11; 14 mg/kg busulfan starting on day -10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day -5 to -2. Fourteen patients received CD34(+)-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft-selected peripheral blood stem cells CD34(+) and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 x 10(5)/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

IL-16 can synergize with early acting cytokines to expand ex vivo CD34+ isolated from cord blood.

We previously reported that interleukin (IL)-16 can induce CD34(+) hematopoietic cells to proliferate and differentiate in vitro into phenotypically and functionally mature dendritic cells. In this study, we investigated the effects of IL-16 on the expansion of CD34(+) cells from human cord blood (CB). CD34(+) CB cells were cultured for 14 days in medium containing a basal cocktail (BC) containing stem cell factor, Flt-3 ligand, thrombopoietin, IL-6, and IL-3 with and without IL-16 as a control. Interleukin-16 added to BC significantly enhanced the expansion of CD34(+) cells (66.47 +/- 1.46-fold vs. 36.23 +/- 1.67-fold), as well as CD34(+)CD38(-) early stem cells (106.67 +/- 2.34-fold vs. 63.42 +/- 1.89-fold) and progenitor cells [colony-forming unit (CFU) -mixed -(GEMM)] and multilineage-committed progenitors [burst-forming unit (BFU-E), CFU-granulocyte, macrophage (-GM), CFU-megakaryocyte (-MK)]. Interleukin-16 also significantly increased long-term culture-initiating cells (160.8 +/- 3.45-fold vs. 83 +/- 2.89-fold with BC alone). Moreover, CD34(+) cells expanded with IL-16 maintained the capacity to differentiate into the lymphoid-B and -NK lineage. The addition of IL-16 to BC increased the migratory capacity of expanded CD34(+) cells compared to BC alone, leaving the expression of CXCR4 unaffected, and decreased the percentage of CD34(+)CD4(+) cells. We showed that IL-16 released endogenously affected the ex vivo expansion of CD34(+) cells. Overall, this study suggests that IL-16 may have a new role in promoting the expansion of hematopoietic stem cells and may represent a new tool for the expansion of CD34(+) cells for clinical applications.

Immunomagnetic selection of progenitor cells from peripheral blood after thawing with an automatic system in a pediatric patient with a neuroblastoma.

BACKGROUND: Immunomagnetic selection of peripheral blood progenitor cells (PBPCs) in patients with tumoral infiltration in marrow makes it possible to reduce contamination of cellular concentrates, but this procedure cannot always be used, mainly because of the low cellular count in apheresis concentrates. STUDY DESIGN AND METHODS: In this case two cellular concentrates taken separately at two different times were selected and cryopreserved; they were thawed with an automatic instrument. RESULTS: After manipulation, a selected concentrate containing 24.16 x 106 CD34+ cells with a purity of 90.15 percent was obtained; vitality after thawing and selection was 88 and 96 percent, respectively. The engraftment was achieved on Day +17 from the infusion of the previously selected PBPCs, as the literature also shows us. CONCLUSION: The time passed between the infusion and the engraftment gives us evidence of the efficacy of immunomagnetic selection carried out after thawing 2 cell units that were collected at different times from the same patient. In this way, it has been possible to perform an autologous transplant in a patient in which CD34+ cells transplant is recommended, but from whom the number of collected cells after a single mobilization cycle would not have been sufficient for the engraftment.

Induction of regulatory T cells after prophylactic treatment with photopheresis in renal transplant recipients.

Extracorporeal photopheresis (ECP), originally used to treat cutaneous T-cell lymphoma, also has been applied to the therapy of transplant rejection. Our aim was to investigate the biologic response in two children who underwent kidney transplantation with ECP as prophylactic treatment. They received conventional immunosuppressive therapy and ECP immediately after transplantation: six applications over the course of 3 weeks. During a 12-month follow-up, the clinical course was favorable in both patients; renal histology was normal 6 months after transplantation. When compared with four transplanted controls, the ECP-treated patients showed lower tumor necrosis factor-alpha serum levels in the short-term and a marked increase of Foxp3-positive T-regulatory cells. T-regulatory cells were still higher than in the controls 1 year after transplantation. These preliminary results suggest that the addition of ECP to standard immunosuppressive therapy induces a tolerogenic shift in the immune system of kidney transplanted patients and may pave the way to preventing chronic rejection.

Chimerism in a child with severe combined immunodeficiency: a case report.

Severe combined immunodeficiency (SCID) represents a group of rare, sometimes fatal, congenital disorders in which there is a combined absence of T-lymphocyte and B-lymphocyte function. Children with SCID die within two years of age, if untreated. The effective treatment for SCID is a hematopoietic stem cell transplantation (HSCT). It has been repeatedly described that in peripheral blood of infants with SCID maternal T cells can be found. Here we report a case of blood chimerism in a one-year-old boy with SCID.

Osteogenesis induced by autologous bone marrow cells transplant in the pediatric skull.

BACKGROUND AND PURPOSE: The ability of cranial bone to repair defects of continuity is limited and it is mostly dependent on the age of the patient. In infancy and in early pediatric age, the scarce thickness of the calvarial bones and the need for a harmonic development of the child's skull limit the application of most of the surgical procedures usually utilized in older patients. We tested the ability of mononucleated cells, derived from the patient's bone marrow and transplanted on the site of the cranial bone defect, to increase the rate of mineralization of the autologous osteogenesis to obtain the complete restoration of the skull continuity. METHOD: Four children, aged 26, 28, 37, and 79 months, respectively, affected by a stabilized and persistent cranial bone defect of posttraumatic or postsurgical origin, were treated. A sandwich-shaped shell, made of extrused absorbable polylactic copolymers material, was used to hold in place a freeze-dried mineralized collagen matrix associated with a nonceramic hydroxyapatite scaffold, where autologous bone marrow mononucleated cells were inseminated. RESULTS: In all patients, a rapid autologous bone osteogenesis was observed with a clear dimensional reduction of the bone defect few months after the autologous bone marrow cells seeding. CONCLUSIONS: The preliminary results of this research suggest the use of autologous bone marrow cells to increase the autologous osteogenesis in early pediatric age in cases in which correction of skull bone defects is best realized with autologous bone.