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Abstract: 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 &y& Elsevier]

The authors compared the efficiency of two different blood cell separators (Amicus and Cobe-Spectra) in collecting peripheral blood progenitor cells for autologous or homologous transplantation. A total number of 129 procedures were performed, 36 with Spectra, 93 with Amicus. There was no difference between Spectra and Amicus efficiencies for CD34+ cell collection (46.685% vs 46.235%; p=n.s) but the platelet efficiencies were 17.31% and 12.54% respectively (p=0.04) and, if autologous and allogeneic collections were considered separately, a marked difference resulted in allogeneic platelet efficiency between 6 Spectra and 23 Amicus procedures (26.83% vs 8.68%, p=0.0004). The authors were able to demonstrate that in 70 Amicus autologous collections there was a different platelet efficiency, if peripheral count was considered: 12 procedures performed with a platelet count > 100 × 109/l had a very low efficiency (6.86%), but this value increased if platelet count lowered (13.02% if between 100 and 50 × 109/l, 22.63% if between 50 and 0 × 109/l, 23 and 35 procedures respectively). The study is preliminary and the number of collections is little, but the overall data suggest that Spectra (AutoPBSC, V 6.0) and Amicus separators have the same efficiency for collecting CD34+ cells while Amicus procedures have a very low platelet contamination, especially with donors. [Copyright &y& Elsevier]

Among the surrounding cells influencing tumor biology, platelets are recognized as novel players as they release microvesicles (MVs) that, once delivered to cancer cells, modulate signaling pathways related to cell growth and dissemination. We have previously shown that physiological delivery of platelet MVs enriched in miR-126 exerted anti-tumor effects in different breast cancer (BC) cell lines. Here, we seek further insight by identifying AKT2 kinase as a novel miR-126-3p direct target, as assessed by bioinformatic analysis and validated by luciferase assay. Both ectopic expression and platelet MV-mediated delivery of miR-126-3p downregulated AKT2 expression, thus suppressing proliferating and invading properties, in either triple negative (BT549 cells) or less aggressive Luminal A (MCF-7 cells) BC subtypes. Accordingly, as shown by bioinformatic analysis, both high miR-126 and low AKT2 levels were associated with favorable long-term prognosis in BC patients. Our results, together with the literature data, indicate that miR-126-3p exerts suppressor activity by specifically targeting components of the PIK3/AKT signaling cascade. Therefore, management of platelet-derived MV production and selective delivery of miR-126-3p to tumor cells may represent a useful tool in multimodal therapeutic approaches in BC patients. [ABSTRACT FROM AUTHOR]

A urine test permits the measure of several urinary markers. This is a non-invasive method for early monitoring of potential kidney damage. In COVID-19 patients, alterations of urinary markers were observed. This review aims to evaluate the utility of urinalysis in predicting the severity of COVID-19. A total of 68 articles obtained from PubMed studies reported that (i) the severity of disease was related to haematuria and proteinuria and that (ii) typical alterations of the urinary sediment were noticed in COVID-19-associated AKI patients. This review emphasizes that urinalysis and microscopic examination support clinicians in diagnosing and predicting COVID-19 severity. [ABSTRACT FROM AUTHOR]

There is a progressive increase in the use of selected hematopoietic progenitor cells after myeloablative therapy in patients affected by malignancies. Our goal was to determine which blood parameters, in the starting cell population, influence the concentration of CD34+ progenitors and the removal of unwanted cells in the final product. Also, we evaluated the hematopoietic recovery and toxicity associated with peripheral blood stem cell infusion. We retrospectively reviewed 53 procedures of positive selection of CD34+ cells, performed with the Ceprate SC immunoadsorption system, in 47 patients affected by various hematologic malignancies and solid tumors. An increased percentage of CD34+ cells in the starting fraction was associated both with the final purity and enrichment of CD34+ cells and with a decreased percentage of CD3+ and CD19+ cells in the final product. A low platelet count before selection had a borderline influence on the recovery of CD34+ cells. Forty patients received a median of 5×106 CD34+ cells per kg; the absolute neutrophil count (ANC) reached 0.5×109/l in a median of 10 days whereas a PLT count above 20×109/l was observed in 14 days. The reinfusion of selected CD34+ cells, containing a very low amount of dymethylsulfoxide, was well tolerated and no adverse reactions were observed. Autologous transplantation with selected CD34+ cells is a safe and well-tolerated procedure in patients affected by hematologic malignancies and solid tumors. Positive selection of CD34+ cells seems to be related to the quality of the apheresis products, particularly to the initial CD34+ cell and PLT content. [Copyright &y& Elsevier]

Platelet- rich plasma (PRP) exhibits regenerative proprieties in wound healing but the biochemical mechanisms are unclear. In this study, autologous PRP with a mean value of 338 × 10 3 platelets/µL was used to treat corneal lesions of different aetiology, while homologous PRP with 1 × 10 6 platelets/µL was used to treat cornel lesions induced by a graft versus host disease. The impact of platelet count on the levels of PDGF AA and BB, VEGF, and EGF in the two PRPs was evaluated after a cycle of freezing/thawing. Treated corneal lesions healed or improved. The levels of PDGF AA and BB, VEGF, and EGF in the autologous PRP raised from 296 ± 61; 201.8 ± 24; 53 ± 14 and 8.9 ± 2 to 1017 ± 253; 924.7 ± 222; 101 ± 46.5 and 174 ± 15.5 pg/mL, while in the homologous PRP were 3.4, 4.5, 3.2 and 2 folds higher, respectively. High level of platelet counts seems not required to treat corneal lesions. [ABSTRACT FROM AUTHOR]

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 ( Nucleo Counter NC-3000, Chemo Metec) 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 Nucleo Counter 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 × 106/ 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 Nucleo Counter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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 (Cl) 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 Cl of extensive chronic GVHD was 6% ± 0.1%. The 1-year Cl of treatment-related mortality was 36% ± 0.3%. After a median follow-up of 18 months, 36 of 80 145%) 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 transplanta- tions 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 urgentlytranspianted. [ABSTRACT FROM AUTHOR]

Abstract: The Italian Group for Bone Marrow Transplantation (Gruppo Italiano Trapianto di Midollo Osseo, GITMO) recently formalized criteria for a shared definition of poor mobilizer in order to facilitate randomized clinical trials and study comparison focusing on the efficacy of current mobilizing regimens. The availability of a standardized tool for poor mobilizer definition suggested us to retrospectively test GITMO criteria feasibility and applicability. Therefore we analyzed medical and laboratory records of adult patients affected by myeloma (MM) or lymphoma undergoing mobilization for autologous peripheral blood HSC collection from January 2010 to June 2011, at Servizio di Emotrasfusione, Istituto di Ematologia, Università Cattolica Del Sacro Cuore, Roma, UOC SIMT AO S. Camillo Forlanini Roma and SIMT Fondazione Policlinico Tor Vergata Roma. We collected data about 227 patients (134 male, 93 female) affected by MM (31.3%) NHL (58.6%) e HD (10.1%). Thirty-nine patients, 21 male and 18 female met proven poor mobilizer criteria definition resulting in a incidence of 17.2% (12.7% in MM, 21.8% in NHL and 4.3% in HD). Eleven patients, seven affected by lymphoma and four affected by myeloma, were defined predicted PM according to major criteria. Eight patients, seven affected by lymphoma and one affected by myeloma, were define predicted PM according to minor criteria. Sixteen out of 39 patients defined as poor mobilizer either according to major or minor criteria underwent collection procedures and eight (20.5%) achieved a cell dose ⩾2×106/kg CD34+ cells. GITMO criteria application was easy and resulted in poor mobilizer incidence comparable to current literature. Definitions of proven poor mobilizer and predicted poor mobilizer according to major criteria were very effective while minor criteria were less predictive. These results came from a retrospective analysis and therefore should be validated in future prospective trial. On the other hand these data could be an early overall view of the foreseeable future of peripheral blood stem cell collection. In conclusion we believe that these criteria will be able to better characterize poor mobilizer phenomenon and, consequently, to identify patients taking advantage from new mobilizing agents. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

The article presents a study on stem cell transplantation in young patients with thalassemia. It mentions that the stem cells were transplanted from haploidentical donors wherein 27 were mothers, two were brothers, and two were fathers. It adds that the transplantation was undertaken through graft processing wherein eight patients received T cell-depleted peripheral blood progenitor cells. Results show that all patients revealed donor chimerism but seven of them rejected their grafts.

To analyze immunohematologic reconstitution, particularly of natural killer (NK) cells, we evaluated 13 β-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. [ABSTRACT FROM AUTHOR]

To investigate an association between KI and WU polyomavirus (KIPyV and WUPyV) infections and CD4+ cell counts, we tested HIV-1-positive patients and blood donors. No association was found between cell counts and virus infections in HIV-1-positive patients. Frequency of KIPyV infection was similar for both groups. WUPyV was more frequent in HIV-1-positive patients. [ABSTRACT FROM AUTHOR]

To investigate an association between KI and WU polyomavirus (KIPyV and WUPyV) infections and CD4+ cell counts, we tested HIV-l-positive patients and blood donors. No association was found between cell counts and virus infections in HIV-l-positive patients. Frequency of KIPyV infection was similar for both groups. WUPyV was more frequent in HIV-l-positive patients. [ABSTRACT FROM AUTHOR]

BACKGROUND: Platelet (PLT) gel has been successfully used in tissue regeneration of diabetic/surgical wounds through the releasing of growth factors such as basic fibroblast growth factor and PLT-derived growth factor. Therefore, the PLT gel could represent a therapeutic tool in treating the deep and painful wounds sometimes occurring during graft-versus-host disease (GVHD). STUDY DESIGN AND METHODS: The aim of this study was to verify the efficacy and safety of PLT gel for treating GVHD ulcers. Allogeneic hemocomponents were used to obtain PLT gel with an automated system for the on-site preparation and application of patient (autologous) or healthy blood donor (allogeneic)-derived fibrin sealant or PLT-rich fibrin (Vivostat system, Vivostat A/S). Six patients with multiple lesions involving dermis (Grade I, n = 2), subcutaneous (Grade II, n = 4), or oral mucosa and related to GVHD underwent PLT gel as local therapy. RESULTS: After the second PLT gel application, the pain disappeared in all cases and the granulation tissue was observed in the four patients with Grade II lesions. After a median of eight PLT gel applications (range, 4-10), five of six patients showed a complete response, while one patient with a partial response died early from multiorgan failure. No side effects were documented. CONCLUSION: These preliminary data show that the PLT gel may be used as a safe and effective tool in the management of mucosal skin lesions related to the GVHD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Nowadays transfusion safety is still put at risk by contamination of pathogens. The Mirasol PRT System blocks the replication of pathogens and white blood cells. Our goal was to quantify the activation of platelets after treatment with the Mirasol device. Materials and methods: From September to December 2013, 131 platelet collections were studied using a simple flow cytometric strategy. Results: There was a significant correlation between the percentage of platelet activated before and after the treatment. Conclusion: Our results induced us to think that the activation of platelets after treatment was acceptable. [Copyright &y& Elsevier]

Breast cancer is one of the most frequent and malignant types of cancer in women, with an increasing morbidity and mortality rate; in particular, treatment of triple negative breast cancer remains a challenge, since the efforts made with targeted therapies were ineffective. Among surrounding cells influencing the biology of cancer cells, platelets are recognizing as novel players. Activated platelets release microvesicles (MVs) that, once delivered to cancer cells, modulate signaling pathways related to cell growth and dissemination; among factors contained in platelet-derived MVs, microRNAs are highly involved in cancer development. The growing interest in ω3 and ω6 polyunsaturated fatty acids (PUFAs) as adjuvants in anti-cancer therapy prompted us to investigate the ability of arachidonic acid (AA) and docosahexaenoic acid (DHA) to modulate MV biological functions. AA induced differential enhancement of platelet-specific microRNAs (miR-223 and miR-126), an effect further enhanced by the presence of DHA. MVs can be delivered to and microRNAs internalized by breast cancer cells, although with different efficiency; analysis of kinetics of MV delivery, indeed, suggested that tumor cells fine-tune the uptake of specific microRNA. Finally, we demonstrated that physiological delivery of platelet miR-223 and miR-126 induced cellular effects in breast cancer cells, including cell cycle arrest, inhibition of migration and sensitivity to cisplatin. These results have been confirmed by exogenous expression of miR-223 and miR-126 through transient transfection experiments. Our preliminary data suggest that ω6/ω3-PUFA supplementation, by modulating microRNA delivery, enhances platelet anti-tumor activities, thus opening new avenues for add-on therapies in cancer patients. [ABSTRACT FROM AUTHOR]

In this work we describe the case report of a woman affected by cancer of the sigmoid colon and with a positive direct antiglobulin test (DAT) and indirect antiglobulin test (IAT). Case report with results: A meticulous medical history showed that the woman had been suffering from recurrent fetal loss. Then she had cardiac and coagulative problems. These data suggested a phospholipid syndrome. Conclusion: The patient had a medical history positive for a phospholipid syndrome and we think that this disease could explain the onset of the autoantibody. [ABSTRACT FROM AUTHOR]

Focuses on a study concerning the potential capability of the Sysmex NE-8000 to detect and characterize the acute of chronic form of lymphatic and myeloid leukaemias. Data on the combination of scattergram 3 and histogram type 5.