ABSTRACT Natural Killer cells are immune leukocytes required for responses against tumor cells and virus-infected cells. In the last decade, natural killer cells have emerged as promising tools in cancer therapy, and clinical studies on patients treated with natural killer cells have revealed increased rates of disease-free survival. In this article, we review results from the major clinical trials that have used natural killer cells for cancer treatment, including their global distribution. We also discuss the major mechanisms of natural killer cell activation and expansion and focus on the advantages and disadvantages of each mechanism for clinical applications. Although natural killer cells can be isolated from several sources, primary natural killer cells are most commonly used in clinical trials. However, the frequency of natural killer cells available in peripheral and cord blood is low, necessitating development of methods for expansion of natural killer cells for clinical use. The development of a platform for the expansion of large-scale good manufacturing practice-compliant natural killer cells has limitations as several methods for natural killer cell activation and expansion yield conflicting results. Only techniques using feeder cells can produce large numbers of cells, allowing the “off-the-shelf” use of natural killer cells. However, advances in cell culture have supported the development of feeder-free platforms for natural killer cell expansion, which is fundamental for improving the safety of this type of cell therapy. virusinfected virus infected decade therapy diseasefree disease free survival article treatment distribution applications sources However low largescale scale practicecompliant practice compliant offtheshelf off shelf “off-the-shelf feederfree

Abstract Introduction Umbilical cord blood is an alternative source of hematopoietic progenitor cells for bone marrow transplantation; however, it is associated with a higher graft failure rate. The presence of a high rate of nucleated red blood cells (NRBCs) seems to be related to a greater capacity for engraftment, although is also associated with fetal distress conditions. We analyzed the correlation of the NRBC with quality parameters and its association with the utilization score of a cord blood unit. Study design and method Data of 3346 units collected in a public cord blood bank from May 2010 to December 2017 were analyzed, retrospectively, to identify factors associated with an increased number of nucleated red blood cells and its correlation with the engraftment capacity measured through total nucleated cells (TNCs) and CD34 positive cells. We also evaluated the utilization score of these units and identified an NRBC cutoff associated with a higher score. Results The median volume collected was 104 mL (42-255), the pre-processing TNC count was 144.77 × 107 (95.46-477.18), the post-processing TNC count was 119.44 × 107 (42.7-477.18), the CD34 count was 4.67 × 106 (0.31-48.01), the NRBC count was 5 (0-202) and the utilization score was 0.0228 (0.00143-0.9740). The NRBC showed a correlation with the collected volume, TNC and CD34 positive cells and a higher utilization score and the receiver operating characteristic (ROC) curve analysis identified the five NRBC/100 leukocytes cutoff that correlates better with the probability of use. No association with pathological conditions and the NRBC rate was observed. Conclusions The NRBC is a feasible parameter for the screening of the cord blood unit (CBU) and the minimum cutoff of five NRBC/100 leukocytes can be a strategy in conjunction with the TNC to identify better units for cord blood bank sustainability. transplantation however NRBCs (NRBCs 334 201 retrospectively TNCs (TNCs CD CD3 10 42255, 42255 42 255 , (42-255) preprocessing pre processing 14477 144 77 144.7 95.46477.18, 954647718 95.46 477.18 95 46 477 18 (95.46-477.18) postprocessing post 11944 119 44 119.4 42.7477.18, 42747718 42.7 7 (42.7-477.18) 467 4 67 4.6 0.3148.01, 0314801 0.31 48.01 0 31 48 01 (0.31-48.01) 0202 202 (0-202 00228 0228 0.022 0.001430.9740. 00014309740 0.00143 0.9740 . 00143 9740 (0.00143-0.9740) ROC (ROC NRBC100 100 NRBC/10 use observed CBU (CBU sustainability 33 20 1 4225 25 (42-255 1447 14 144. 46477 95.46477.18 95464771 9546 95.4 47718 477.1 9 47 (95.46-477.18 1194 11 119. 7477 42.7477.18 4274771 427 42. (42.7-477.18 6 4. 3148 0.3148.01 031480 031 0.3 4801 48.0 3 (0.31-48.01 020 (0-20 0022 022 0.02 001430 0.001430.9740 0001430974 000143 0.0014 09740 0.974 0014 974 (0.00143-0.9740 NRBC10 NRBC/1 2 422 (42-25 4647 95.46477.1 9546477 954 95. 4771 477. (95.46-477.1 747 42.7477.1 427477 (42.7-477.1 314 0.3148.0 03148 03 0. 480 48. (0.31-48.0 02 (0-2 002 0.0 0.001430.974 000143097 00014 0.001 0974 0.97 001 97 (0.00143-0.974 NRBC1 NRBC/ (42-2 464 95.46477. 954647 (95.46-477. 74 42.7477. 42747 (42.7-477. 0.3148. 0314 (0.31-48. (0- 00 0.001430.97 00014309 0001 0.00 097 0.9 (0.00143-0.97 (42- 95.46477 95464 (95.46-477 42.7477 4274 (42.7-477 0.3148 (0.31-48 (0 0.001430.9 0001430 000 09 (0.00143-0.9 (42 95.4647 (95.46-47 42.747 (42.7-47 0.314 (0.31-4 ( 0.001430. (0.00143-0. (4 95.464 (95.46-4 42.74 (42.7-4 (0.31- 0.001430 (0.00143-0 (95.46- (42.7- (0.31 (0.00143- (95.46 (42.7 (0.3 (0.00143 (95.4 (42. (0. (0.0014 (95. (0.001 (95 (0.00 (9 (0.0

ABSTRACT Objective Phase 1 clinical trial to determine feasibility, safety, and efficacy of a new advanced cell therapy product for treatment of knee articular cartilage injuries. Methods Three participants with knee focal chondral lesions were included, with no signs of osteoarthritis. Chondrocytes were obtained through knee arthroscopy, cultured in collagen membrane for 3 weeks at the laboratory, subjected to tests to release the cell therapy product, and implanted. All patients underwent a specific 3-month rehabilitation protocol, followed by assessments using functional and imaging scales. The main outcome was the incidence of severe adverse events. Results Three participants were included and completed the 2-year follow-up. There was one severe adverse event, venous thrombosis of distal leg veins, which was no associated with therapy, was treated and left no sequelae. The clinical and radiological scales showed improvement in the three cases. Conclusion The preliminary results, obtained with the described methodology, allow concluding that this product of advanced cell therapy is safe and feasible. ReBEC platform registration number: RBR-6fgy76

ABSTRACT Objective The objective of the present study is to evaluate the association of red blood cell distribution width with acute kidney injury in sepsis. Methods This is a retrospective study of 849 critically ill patients with sepsis in intensive care unit. Demographic data, renal function, inflammation, complete blood count, and acid-base parameters were compared between acute kidney injury and non-acute kidney injury groups. Therefore, a multivariate analysis was performed to observe independent predictive factors. Results Comparatively, higher levels of C-reactive protein, lactate, red blood cell distribution width, and Simplified Acute Physiology Score 3 were found in the acute kidney injury group. The study showed a higher frequency of women, hemoglobin (Hgb) concentration, platelets, bicarbonate and PaO2/FiO2 ratio in the non-acute kidney injury group. In addition, there was an independent association of comorbidity-chronic kidney disease [OR 3.549, 95%CI: 1.627-7.743; p<0.001], urea [OR 1.047, 95%CI: 1.036-1.058; p<0.001] and RDW [OR 1.158, 95%CI: 1.045-1.283; p=0.005] with acute kidney injury in sepsis patients. Conclusion As an elective risk factor, red blood cell distribution width was independently associated with sepsis-related acute kidney injury. Thus, red blood cell distribution width acts like a predictive factor for sepsis-induced acute kidney injury in intensive care unit admission.

ABSTRACT To describe a case of autologous chondrocyte implantation after cell culture contamination by Mycoplasma pneumoniae and the measures taken to successfully complete cell therapy in a patient with focal chondral lesion. A 45-year-old male patient, complaining of chronic pain on the knee and no history of trauma. He had a chondral lesion in the trochlear region of the femur and clinical tests compatible with pain in the anterior compartment of the knee. Conservative treatment failed to alleviate symptoms. Surgical treatment was indicated, but due to the size of the lesion, membrane-assisted autologous chondrocyte implantation was the technique of choice. Cartilage biopsies were collected from the intercondylar region of the distal femur. After isolation, chondrocytes were expanded ex vivo in a trained laboratory, for three weeks, and seeded onto a commercially available collagen membrane prior to implantation in the knee. Two days before surgery, a cell culture sample tested positive for Mycoplasma pneumoniae. The source of contamination was found to be autologous blood serum, extracted from the patient´s peripheral vein, and used to supplement the cell culture medium. After treating the patient with antibiotics, all procedures were repeated and the new final cell product, free from contaminants, was successfully implanted. We discuss the strategies available to deal with this situation, and describe the results of this particular case, which led to modifications in the autologous chondrocyte implant protocol.

A púrpura trombocitopênica neonatal aloimune é uma doença grave, na qual a mãe produz anticorpos contra antígenos plaquetários fetais herdados do pai, e é ainda subdiagnosticada na prática clínica. É considerada o equivalente plaquetário da doença hemolítica do recém-nascido, com a diferença que o primeiro filho é afetado, apresentando trombocitopenia fetal e/ou neonatal. Há risco significativo de hemorragia intracraniana e sequelas neurológicas graves, com tendência a trombocitopenia mais grave e mais precoce nas gestações subsequentes. Este artigo relata um caso de trombocitopenia aloimune neonatal na segunda gestação afetada e discute diagnóstico, manejo e importância clínica dessa doença na prática clínica.

Neonatal alloimmune thrombocytopenia is a serious disease, in which the mother produces antibodies against fetal platelet antigens inherited from the father; it is still an underdiagnosed disease. This disease is considered the platelet counterpart of the RhD hemolytic disease of the fetus and newborn, yet in neonatal alloimmune thrombocytopenia the first child is affected with fetal and/or neonatal thrombocytopenia. There is a significant risk of intracranial hemorrhage and severe neurological impairment, with a tendency for earlier and more severe thrombocytopenia in subsequent pregnancies. This article reports a case of neonatal alloimmune thrombocytopenia in the second pregnancy affected and discusses diagnosis, management and the clinical importance of this disease.

OBJETIVO: Comparar as células-tronco mesenquimais humanas obtidas de filtros de coleta reutilizáveis àquelas coletadas em filtros descartáveis e caracterizá-las utilizando os critérios da International Society for Cellular Therapy. MÉTODOS: Foram isoladas células-tronco mesenquimais humanas de kits de coleta de medula óssea reutilizáveis e descartáveis, pela lavagem dos filtros com meio de cultura. As células isoladas foram caracterizadas de acordo com os critérios estabelecidos pela International Society for Cellular Therapy, por meio das técnicas de citometria de fluxo, diferenciação in vitro e citoquímica. RESULTADOS: As amostras foram obtidas de filtro descartável (n=3) e reutilizável (n=3). Todas as amostras obtidas de filtros descartáveis produziram células-tronco mesenquimais, e todas as células-tronco mesenquimais humanas derivadas de medula óssea preencheram os critérios estabelecidos pela International Society for Cellular Therapy. CONCLUSÃO: Este estudo mostrou que as células-tronco mesenquimais também podem ser obtidas de kits de coleta reutilizáveis (que permanecem em uso em vários centros, no mundo inteiro), para serem empregadas em pesquisa como uma fonte alternativa e ética.

OBJECTIVE: To compare human mesenchymal stem cells obtained from reusable and disposable filters and to characterize them according to the criteria of the International Society of Cellular Therapy. METHODS: Human mesenchymal stem cells were isolated from bone marrow collection reusable sets and compared with those obtained from disposable sets by washing the filters with cell culture media. The isolated cells were characterized according to the criteria of the International Society of Cellular Therapy using flow cytometry, differentiation in vitro, and cytochemistry techniques. RESULTS: Samples were obtained from disposable (n=3) and from reusable collection sets (n=3). All samples obtained from bone marrow disposable sets successfully produced mesenchymal stem cells. All bone marrow derived mesenchymal stem cells were characterized and fulfilled the criteria established by International Society of Cellular Therapy. CONCLUSION: This study showed that mesenchymal stem cells can also be obtained from reusable collection sets (which are still used in several centers around the world) to be employed in research as an alternative and ethical source.

Objetivo: Avaliar fatores que afetam o rendimento da coleta em pacientes submetidos à leucaférese de grande volume para obtenção de células-tronco hematopoiéticas do sangue periférico para autotransplante. Métodos: Análise retrospectiva de 304 doadores de células-tronco hematopoiéticas de sangue periférico para autotransplante submetidos à mobilização com fator de crescimento hematopoiético (geralmente G-CSF), associado ou não à quimioterapia, no Hospital Israelita Albert Einstein, de Fevereiro de 1999 a Junho de 2010. O objetivo da coleta foi obter pelo menos 2 × 106 CD34+ células/kg peso. Os fatores pré-mobilização incluíam idade, sexo e diagnóstico do paciente. Os parâmetros pós-mobilização avaliados foram contagem de leucócitos, células imaturas, células mononucleares, células CD34+, plaquetas e nível de hemoglobina no sangue periférico. O efeito desses fatores no rendimento da coleta de CTH foi investigado por meio de regressão logística (análise univariada e multivariada). Resultados: A análise univariada revelou os seguintes fatores pré-mobilização estatisticamente significantes: diagnóstico de leucemia mieloide aguda (p = 0,017) e outras doenças hematológicas (p = 0,023), células imaturas circulantes (p = 0,001), granulócitos (p = 0,002), nível de hemoglobina (p = 0,016) e contagem de células CD34+ no sangue periférico (p < 0,001) no primeiro dia de coleta. Entretanto, só a contagem de células CD34+ no sangue periférico manteve-se associada de forma significante ao rendimento ruim da coleta de células-tronco hematopoiéticas na análise multivariada. Conclusão: Neste estudo, a contagem de células CD34+ no sangue periférico foi o único fator significantemente associado ao rendimento da coleta de células-tronco hematopoiéticas com leucaférese de grande volume para autotransplante.

Objective: To evaluate factors affecting peripheral blood hematopoietic stem cell yield in patients undergoing large-volume leukapheresis for autologous peripheral blood stem cell collection. Methods: Data from 304 consecutive autologous peripheral blood stem cell donors mobilized with hematopoietic growth factor (usually G-CSF), associated or not with chemotherapy, at Hospital Israelita Albert Einstein between February 1999 and June 2010 were retrospectively analyzed. The objective was to obtain at least 2 × 106 CD34+ cells/kg of body weight. Pre-mobilization factors analyzed included patient's age, gender and diagnosis. Post mobilization parameters evaluated were pre-apheresis peripheral white blood cell count, immature circulating cell count, mononuclear cell count, peripheral blood CD34+ cell count, platelet count, and hemoglobin level. The effect of pre and post-mobilization factors on hematopoietic stem cell collection yield was investigated using logistic regression analysis (univariate and multivariate approaches). Results: Pre-mobilization factors correlating to poor CD34 + cell yield in univariate analysis were acute myeloid leukemia (p = 0.017) and other hematological diseases (p = 0.023). Significant post-mobilization factors included peripheral blood immature circulating cells (p = 0.001), granulocytes (p = 0.002), hemoglobin level (p = 0.016), and CD34+ cell concentration (p < 0.001) in the first harvesting day. However, according to multivariate analysis, peripheral blood CD34+ cell content (p < 0.001) was the only independent factor that significantly correlated to poor hematopoietic stem cell yield. Conclusion: In this study, peripheral blood CD34+ cell concentration was the only factor significantly correlated to yield in patients submitted to for autologous collection.