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Background and Aim: A complex relationship between immune system and metabolic pathway exists and can induce oxidative stress. The objective of this study was to design a new methodology allowing the measurement of oxidative status of leukocytes., Methods and Results: We developed a flow cytometry technique, based on C11-BODIPY 581/591 staining, to evaluate peroxidation in leukocytes. We defined the Peroxidation of Leukocytes Index Ratio (PLIR) as the ratio between the damage after AAPH-induced and PMA-induced peroxidation, using Trolox as standard antioxidant. Sensitivity of the method was assessed by correlating results with plasma antioxidant capacity (TRAP and FRAP), levels of endogenous antioxidants (uric acid and sulfhydryls) and markers of metabolic status (cholesterol, triglycerides, glucose and insulin). PLIR measures the ratio between the resistance to exogenous and endogenous ROS injury, independently from baseline level of oxidation, which was directly correlated with plasma cholesterol on lymphocytes (0.738, p=0.029), monocytes (0.691, p=0.047) and neutrophils (0.690, p=0.047). PLIR of lymphocytes was inversely correlated with uric acid (-0.810, p=0.009) and FRAP (-0.738, p=0.029) levels. On the other hand, PLIR of monocytes was directly correlated with the total scavenger antioxidant capacity attributable to nutritional antioxidants (0.738, p=0.029), calculated as the difference between TRAP and the contribution of uric acid and sulfhydryls to its value., Conclusions: This study reports a feasible and reproducible new flow cytometry assay for assessing the leukocytes redox status. PLIR discriminates between reducing and scavenger activities and is able to appreciate the potentially dangerous effect of uric acid on innate immune response., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Unlabelled: Eighty patients with high-risk hematologic malignancies underwent unmanipulated, G-CSF–primed BM transplantation from an haploidentical family donor. Patients were transplanted in first or second complete remission (CR, standard-risk: n =45) or in > second CR or active disease (high-risk: n =35). The same regimen for GVHD prophylaxis was used in all cases. The cumulative incidence (CI) of neutrophil engraftment was 93% 0.1%. The 100-day CIs for II-IV and III-IV grade of acute GVHD were 24% 0.2% and 5% 0.6%, respectively. The 2-year CI of extensive chronic GVHD was 6% 0.1%. The 1-year CI of treatment-related mortality was 36% 0.3%. After a median follow-up of 18 months, 36 of 80 (45%) patients are alive in CR. The 3-year probability of overall and disease-free survival for standard-risk and high-risk patients was 54% 8% and 33% 9% and 44% 8% and 30% 9%, respectively. In multivariate analysis, disease-free survival was significantly better for patients who had standard-risk disease and received transplantations after 2007. We conclude that unmanipulated, G-CSF–primed BM transplantation from haploidentical family donor provides very encouraging results in terms of engraftment rate, incidence of GVHD and survival and represents a feasible, valid alternative for patients with high-risk malignant hematologic diseases, lacking an HLA identical sibling and in need to be urgently transplanted., Key Points: Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation. Haploidentical hematopoietic stem cell transplantation for hematologic malignancies.

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.

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.