Characterization and Clinical Assessment of a Peculiar Case of Hemolytic Anemia.

Thalassemic diseases are characterized by a reduced (ß+) or absent (ß0) synthesis of the globin chains of hemoglobin (Hb) due to genetic mutations. ß-thalassemia was more frequent in the Mediterranean area, but now it is diffused worldwide. Three possible genetic forms can be distinguished: ß0/ß0, the most severe (Cooley's disease); ß0/ß+ of intermediate severity; ß+/ß+ associated with ß-thalassemia intermedia or minor. Recently, a clinical non-genetic classification has been proposed: transfusion-dependent thalassemia (TDT), requiring regular lifetime blood transfusions, and non-transfusion-dependent thalassemia (NTDT), requiring occasional transfusions to manage acute cases. In this report, we studied a patient whose blood count indicated a severe anemia but also showed thrombocytosis, leukocytosis, and an elevated number of nucleated red blood cells (NRBC). These altered blood parameters suggested initially a possible diagnosis of hemoglobinopathy or myeloproliferative syndrome. The molecular and genetic analyses demonstrated the presence of HbF (5.3%) and HbA2 (7.7%) and the presence of the homozygote mutation (IVS1.6T>C) in the ß-globin gene. According to these data, a diagnosis of ß-thalassemia intermedia form has been proposed. Nevertheless, the clinical condition, the presence of thrombocytosis, leukocytosis, an elevated number of NRBC, and the frequent blood transfusions lead to reclassification of the patient as TDT subject. Consequently, this result suggests that a unique genotype-phenotype correlation is not possible in the presence of ß+mutations since other concomitant pathologies can exacerbate the disease.

Urine Parameters in Patients with COVID-19 Infection.

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.

Platelet-Derived miR-126-3p Directly Targets AKT2 and Exerts Anti-Tumor Effects in Breast Cancer Cells: Further Insights in Platelet-Cancer Interplay.

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.

Matched-Pair Analysis of Transplant from Haploidentical, Unmanipulated Bone Marrow Donor versus HLA Identical Sibling for Patients with Hematologic Malignancies.

A matched-pair analysis of transplant-related outcomes was carried out in 116 of 255 consecutive patients who received transplants from an HLA identical sibling (n = 58) or haploidentical related donor (n = 58). The 2 patient series were matched with 9 variables: period of transplant, patient and donor age, sex, diagnosis, disease phase, conditioning regimen, donor-recipient sex, and cytomegalovirus (CMV) status combinations. As graft-versus-host disease (GVHD) prophylaxis, all patients received the standard cyclosporine and methotrexate association with the addition of anti-thymocyte globulins, mycophenolate mofetil, and basiliximab in haploidentical, unmanipulated bone marrow recipients. Anti-infectious management, transfusion policy, and supportive care were identical for all patients. By comparing the 2 patient series, no statistically significant difference was observed for the cumulative incidence of advanced acute and extensive chronic GVHD, transplant-related mortality, and relapse. With a median follow-up of 3.5 years, the 5-year disease-free survival was 37% ± 6% and 36% ± 6% for HLA identical sibling and haploidentical recipients, respectively. The results of transplant from HLA identical siblings and haploidentical donors are comparable. Regardless of the HLA matching, other factors known to affect the transplant outcomes, such as donor-recipient age, sex, and CMV status combinations, might drive the search for the best donor.

Polyunsaturated fatty acids modulate the delivery of platelet microvesicle-derived microRNAs into human breast cancer cell lines.

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.

In Vitro Exposure to Prostratin but Not Bryostatin-1 Improves Natural Killer Cell Functions Including Killing of CD4+ T Cells Harboring Reactivated Human Immunodeficiency Virus.

In the attempt of purging the HIV-1 reservoir through the "shock-and-kill" strategy, it is important to select latency-reversing agents (LRAs) devoid of deleterious effects on the antiviral function of immune effector cells. Here, we investigated two LRAs with PKC agonist activity, prostratin (PRO) and bryostatin-1 (BRY), for their impact on the function of natural killer (NK) cells, the major effectors of innate immunity whose potential in HIV-1 eradication has emerged in recent clinical trials. Using NK cells of healthy donors, we found that exposure to either PRO or BRY potently activated NK cells, resulting in upmodulation of NKG2D and NKp44 activating receptors and matrix metalloprotease-mediated shedding of CD16 receptor. Despite PRO and BRY affected NK cell phenotype in the same manner, their impact on NK cell function was diverse and showed considerable donor-to-donor variation. Altogether, in most tested donors, the natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells were either improved or maintained by PRO, while both activities were impaired by BRY. Moreover, we analyzed the effect of these drugs on the capacity of treated NK cells to kill autologous latently infected CD4+ T cells reactivated via the same treatment. First, we found that PRO but not BRY increased upmodulation of the ULBP2 ligand for NKG2D on reactivated p24+ cells. Importantly, we showed that clearance of reactivated p24+ cells by NK cells was enhanced when both targets and effectors were exposed to PRO but not to BRY. Overall, PRO had a superior potential compared with BRY as to the impact on key NK cell functions and on NK-cell-mediated clearance of the HIV-1 reservoir. Our results emphasize the importance of considering the effects on NK cells of candidate "shock-and-kill" interventions. With respect to combinative approaches, the impact on NK cells of each LRA should be re-evaluated upon combination with a second LRA, which may have analogous or opposite effects, or with immunotherapy targeting NK cells. In addition, avoiding co-administration of LRAs that negatively impact ADCC activity by NK cells might be essential for successful application of antibodies or vaccination to "shock-and-kill" strategies.

Haploidentical HSCT for hemoglobinopathies: improved outcomes with TCRαß+/CD19+-depleted grafts.

We examined outcomes of haploidentical hematopoietic cell transplantation (haplo-HCT) using T-cell receptor αß+ (TCRαß+)/CD19+-depleted grafts (TCR group, 14 patients) in children with hemoglobinopathies. Patients received a preparative regimen consisting of busulfan, thiotepa, cyclophosphamide, and antithymocyte globulin preceded by fludarabine, hydroxyurea, and azathioprine. The median follow-up among surviving patients was 3.9 years. The 5-year probabilities of overall survival (OS) and disease-free survival (DFS) were 84% and 69%, respectively. The incidence of graft failure was 14%. We compared outcomes to a historical group of 40 patients with hemoglobinopathies who received CD34+-selected grafts (CD34 group). The median follow-up of surviving patients for the CD34 group was 7.5 years. The 5-year probabilities of OS and DFS were 78% and 39%, respectively. The CD34 group had a significantly higher incidence of graft failure (45%) than the TCR group (14%) (P = .048). The incidences of grades 2 to 4 acute graft-versus-host disease (GVHD) in the TCR and CD34 groups were 28% and 29%, respectively, and 21% and 10% (P = .1), respectively, for extensive chronic GVHD. Viral reactivation was common in both groups. The overall incidence of posttransplant lymphoproliferative disorders for the entire group was 16%. Among all patients, 5 developed autoimmune hemolytic anemia or thrombocytopenia, with the overall cumulative incidence of 11%. The 2 groups showed suboptimal CD4+ recovery within the first 6 months of transplantation with no significant difference between groups. These data demonstrate that TCRαß+/CD19+-depleted grafts are associated with a reduced incidence of graft failure, but delayed immune reconstitution and associated morbidity and mortality remain a significant challenge.

Platelet-rich plasma as treatment for persistent ocular epithelial defects.

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.

Cancer of the sigmoid colon and antibodies. A puzzle.

UNLABELLED: 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.

Apheresis in the treatment of recalcitrant atopic dermatitis: case series and review of the literature.

BACKGROUND: Atopic dermatitis is a chronic disabling inflammatory skin disorder, typically characterized by intensely itching, oozing, crusted, eroded vesicles or papules developing on erythematous plaques. Conventional treatments, both topical and systemic, may produce unsuccessful and unsatisfactory results. OBJECTIVES: we aimed to assess the efficacy of apheretic treatments in patients with severe, recalcitrant AD, in particular, the pruritic component. PATIENTS AND METHODS: four patients affected by recalcitrant and debilitating atopic dermatitis, who had previously received conventional topical and systemic therapies with poor clinical improvement, were treated with extracorporeal photopheresis or therapeutic plasma exchange. RESULTS: a satisfactory response to apheresis was observed with a reduction of pruritus and skin lesions. CONCLUSION: In our experience, apheretic therapies might be used as monotherapy but, more effectively, in combination with topical and/or systemic treatments. Indeed, they proved to be a safe "enhancer" for increasing the efficacy of conventional therapeutics.

Platelet activation by riboflavin and UV light: is it really the other side of the coin?

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.

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.

A new flow cytometry method to measure oxidative status: the Peroxidation of Leukocytes Index Ratio (PLIR).

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.

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.

Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation for patients with high-risk hematologic malignancies.

UNLABELLED: Eighty patients with high-risk hematologic malignancies underwent unmanipulated, G-CSF­primed 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-CSF­primed 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.

Poor mobilizer: a retrospective study on proven and predicted incidence according to GITMO criteria.

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 ⩾2×10(6)/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.