Transcriptome profiling reveals distinct alterations in the B-cell signature and dysregulation of peripheral B-cell subsets in sickle cell anemia patients.

Sickle cell anemia (SCA) is characterized by immune system activation and heightened susceptibility to infections. We hypothesized that SCA patients exhibit transcriptional alterations in B-cell-related genes, impacting their peripheral B-cell compartment and leading to dysregulated humoral immunity and increased infection susceptibility. Our objective was to conduct an in silico analysis of whole blood transcriptomes from SCA patients and healthy controls obtained from public repositories. We aimed to identify alterations in the adaptive immune system and validate these findings in our own SCA patient cohort. Bioinformatic analyses unveiled significant transcriptional alterations in B-cell signatures, developmental pathways, and signaling pathways. These results were validated in peripheral blood mononuclear cells from our SCA patient cohort and controls using real-time polymerase chain reaction and flow cytometry. Ninety genes exhibited differential expression, with 70 upregulated and 20 downregulated. Dysregulation in the B-cell compartment of SCA patients was evident, characterized by increased frequencies of immature and naive B-cells, and decreased percentages of memory B-cell subsets compared with healthy controls. Our findings highlight previously unexplored transcriptional and quantitative alterations in peripheral B-cells among SCA patients. Understanding these changes sheds light on the mechanisms contributing to the heightened infection risk in this population. Future studies should delve deeper into these molecular changes to develop targeted interventions and therapeutic strategies aimed at mitigating infection susceptibility in individuals with SCA.

Consensus of the Brazilian association of hematology, hemotherapy and cellular therapy on patient blood management: Anemia tolerance.

Anemia is a pathological condition in which the hemoglobin and red blood cell mass decrease; it is mainly defined by the concentration of hemoglobin in the blood. The World Health Organization guidelines establish specific values to define anemia in different population groups. Early detection of anemia can also be a valuable indicator of underlying medical conditions. Clinical studies have explored the relationship between perioperative anemia and morbidity, highlighting the need for more judicious therapeutic strategies, such as the use of Patient Blood Management, which aims to prevent and treat anemia in a personalized and effective way. Patient Blood Management emerges as a promising approach to dealing with anemia, recognizing that its correction through transfusion always carries risks and that personalized prevention and treatment can offer better outcomes for patients.

Consensus of the Brazilian association of hematology, hemotherapy and cellular therapy on patient blood management: Anemia tolerance mechanisms.

Understanding the physiological concepts of oxygen delivery is essential to discern the mechanisms that influence its increase, reduction or maintenance in the body. This text explores the different mechanisms that help maintain oxygen delivery even in the face of reduced hemoglobin levels. Adequate oxygen delivery ensures tissue and metabolic balance, which is crucial to avoid harmful consequences such as metabolic acidosis and cellular dysoxia. The complex interaction between variables such as cardiac output, hemoglobin and heart rate (HR) plays a fundamental role in maintaining oxygen delivery, allowing the body to temporarily adjust to situations of anemia or high metabolic demand. It is important to emphasize that blood transfusions should not be based on fixed values, but rather on individual metabolic needs. Strategies to reduce myocardial consumption and monitor macro and micro hemodynamics help in making rational decisions. Individualizing treatment and considering factors such as blood viscosity in relation to the benefits of transfusion are increasingly relevant to optimize therapy and minimize risks, especially in complex clinical scenarios, such as neurocritical patients and trauma victims.

Consensus of the Brazilian association of hematology, hemotherapy and cellular therapy on patient blood management: Assessment and management of postoperative anemia.

Postoperative anemia is a complex clinical issue that requires attention due to its ramifications on the patient's recovery and prognosis. Originating from multiple determinants, such as intraoperative blood loss, hemolysis, nutritional deficiencies, systemic inflammation and impact on the bone marrow, postoperative anemia has varied and often challenging presentations. Patients undergoing major surgical procedures, in particular, are susceptible to developing anemia due to the considerable associated blood loss. Accurate diagnosis plays a crucial role in the approach, requiring meticulous hematological analysis, including hemoglobin, hematocrit and reticulocyte count, as well as an in-depth investigation of the underlying causes. An additional challenge arises in the form of the excessive practice of phlebotomy during hospitalization for clinical monitoring. Although it is essential to assess the progression of anemia, frequent removal of blood may contribute to iatrogenic anemia, further delaying recovery and possibly increasing susceptibility to infection.

Influence of Different Photobiomodulation Parameters on Multi-Potent Adipose Tissue Mesenchymal Cells In Vitro.

Objective: Investigating the effect of different parameters of photobiomodulation (PBM) with low-power laser on multi-potent mesenchymal stem cells (MSCs) derived from adipose tissue in terms of proliferation and cell death. Methods: MSCs were submitted to PBM applications with combinations of the following physical parameters: control group (no intervention), wavelengths of 660 and 830 nm; energy of 0.5, 2, and 4 J; and power of 40 and 100 mW. MSC analysis was performed using MetaXpress® software at 24, 48, and 72 h. Results: Irradiation promoted a significant increase in cell proliferation (p < 0.05), with 830 nm laser, 100 mW, with energy of 0.5, 2, and 4 J in relation to the control group at all times. PBM with 660 nm, power of 40 mW, and energy of 0.5, 2, and 4 J produced greater cell death at 24 h compared with the control group. At the time of 72 h, there was no significant difference concerning cell death. Conclusions: According to the results found, we can conclude that both wavelengths were effective; however, the 830 nm laser was more effective in terms of cell proliferation compared with the 660 nm laser. The 660 nm wavelength showed a significant increase in cell death when compared with the 830 nm laser.

Umbilical Cord Mesenchymal Stromal Cells for Steroid-Refractory Acute Graft-versus-Host Disease.

BACKGROUND: Steroid-refractory acute graft-vs.-host disease (SR-aGVHD) is a complication of allogeneic hematopoietic stem cell transplantation with a dismal prognosis and for which there is no consensus-based second-line therapy. Ruxolitinib is not easily accessible in many countries. A possible therapy is the administration of mesenchymal stromal cells (MSCs). METHODS: In this retrospective study, 52 patients with severe SR-aGVHD were treated with MSCs from umbilical cord (UC-MSCs) in nine institutions. RESULTS: The median (range) age was 12.5 (0.3-65) years and the mean ± SD dose (×106/kg) was 4.73 ± 1.3 per infusion (median of four infusions). Overall (OR) and complete response (CR) rates on day 28 were 63.5% and 36.6%, respectively. Children (n = 35) had better OR (71.5% vs. 47.1%, p = 0.12), CR (48.6% vs. 11.8%, p = 0.03), overall survival (p = 0.0006), and relapse-free survival (p = 0.0014) than adults (n = 17). Acute adverse events (all of them mild or moderate) were detected in 32.7% of patients, with no significant difference in children and adult groups (p = 1.0). CONCLUSIONS: UC-MSCs are a feasible alternative therapy for SR-aGVHD, especially in children. The safety profile is favorable.

Outcomes of thrombotic thrombocytopenic purpura patients submitted to therapeutic plasma exchange in a single center in Brazil.

INTRODUCTION: Immune thrombotic thrombocytopenic purpura (iTTP) is characterized by acute systemic microvascular thrombosis and is associated with a high morbidity and mortality, especially in delayed diagnosis (later than 6-7 days from symptoms). iTTP data in Brazil is scarce, so we aimed to characterize the clinical presentation and identify predictors of death risk in patients with this disease in Brazil. METHODS: In this single-center retrospective study the patients who underwent therapeutic plasma exchange (TPE) for presumptive or confirmed iTTP were evaluated regarding the epidemiological, clinical, laboratorial characteristics and management. RESULTS: A total of 50 patients (90 % female), with median age (IQR) of 34.1 (27-47) years, were enrolled, of which 12 (24 %) died. The most frequent symptoms were neurological (96 %), bleeding (76 %), gastrointestinal (52 %), fever (38 %), and cardiovascular (22 %). Neurological focal deficit and cardiovascular symptoms were more frequently observed in the non-survivor group (P = 0.0019 and P = 0.007, respectively). The mean ± SD number of days from beginning of symptoms to first TPE was 12.22 ± 7.91. We identified an association regarding mortality rate with a score MITS ≥ 2 points (P = 0.04), a higher indirect bilirubin (P = 0.0006), a higher number of transfused red blood cell units (P = 0.025), and platelet transfusion (P = 0.027). CONCLUSION: Delayed diagnosis appears to be associated with a higher frequency of neurological symptoms and mortality. Intensity of hemolysis and signs of organ ischemia, such as cardiovascular symptoms and focal neurological deficit, are indicators of death risk.

Allogeneic haematopoietic stem cell transplantation resets T- and B-cell compartments in sickle cell disease patients.

Objectives: Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is the only currently available curative treatment for sickle cell disease (SCD). Here, we comprehensively evaluated the reconstitution of T- and B-cell compartments in 29 SCD patients treated with allo-HSCT and how it correlated with the development of acute graft-versus-host disease (aGvHD). Methods: T-cell neogenesis was assessed by quantification of signal-joint and ß-chain TCR excision circles. B-cell neogenesis was evaluated by quantification of signal-joint and coding-joint K-chain recombination excision circles. T- and B-cell peripheral subset numbers were assessed by flow cytometry. Results: Before allo-HSCT (baseline), T-cell neogenesis was normal in SCD patients compared with age-, gender- and ethnicity-matched healthy controls. Following allo-HSCT, T-cell neogenesis declined but was fully restored to healthy control levels at one year post-transplantation. Peripheral T-cell subset counts were fully restored only at 24 months post-transplantation. Occurrence of acute graft-versus-host disease (aGvHD) transiently affected T- and B-cell neogenesis and overall reconstitution of T- and B-cell peripheral subsets. B-cell neogenesis was significantly higher in SCD patients at baseline than in healthy controls, remaining high throughout the follow-up after allo-HSCT. Notably, after transplantation SCD patients showed increased frequencies of IL-10-producing B-regulatory cells and IgM+ memory B-cell subsets compared with baseline levels and with healthy controls. Conclusion: Our findings revealed that the T- and B-cell compartments were normally reconstituted in SCD patients after allo-HSCT. In addition, the increase of IL-10-producing B-regulatory cells may contribute to improve immune regulation and homeostasis after transplantation.

Neutrophil-to-lymphocyte ratio and D-dimer are biomarkers of death risk in severe COVID-19: A retrospective observational study.

BACKGROUND AND AIMS: Over 4 million deaths from coronavirus disease (COVID)-19 have been reported in the world. Several biomarkers have been identified that predict disease severity, but there is still a need to identify biomarkers for death risk in severe COVID-19. We aim to define amongst the biomarkers already identified those which are mostly associated with increased death rate in patients with severe COVID-19. METHODS: In this retrospective study conducted in three public hospitals linked to the Medical School of Ribeirão Preto, Brazil, patients with severe COVID-19 were evaluated regarding biomarkers (neutrophil-to-lymphocyte ratio-NLR, D-dimer, fibrinogen) of death risk, obtained before administration of corticosteroids. RESULTS: Thirty-nine (32.8%) of the 119 patients included (104 [87.4%] on mechanical ventilation) died during hospitalization. Non-survivor group had higher median (range) NLR (12.63 [2.6-115] vs 7.43 [0.43-31.8]; P = .001), D-dimer (2.17 [0.27-20.00] vs 1.57 [0.28-20.00]; P = .03), but lower fibrinogen (631 [353-1078] vs 705 [407-1200]; P = .02). The group with NLR ≥ 10 and D-dimer ≥ 2 µg/mL had a higher death risk than the group with NLR < 10 and D-dimer < 2 µg/mL (OR: 5.39; CI 95%: 1.5-19.42; P = .01). CONCLUSION: High NLR and D-dimer, especially when combined, are predictors of death risk for patients with severe COVID-19 and should be incorporated into their evaluation.

COVID-19 Infection in Sickle Cell Patients in a Developing Country: A Case Series.

Sickle cell disease is characterized by vaso-occlusive phenomena and haemolytic anaemia. There is a significant concern that the overlap of COVID-19 lung disease with acute chest syndrome that occurs in sickle cell patients may result in serious complications. Case reports of sickle cell patients with COVID-19 have been published. Here, we present a case series of COVID-19 infection in sickle cell patients in a developing country (Brazil). Only 10 patients tested positive so far for SARS-CoV-2 of 600 patients followed at our institution, of which 8 needed hospitalization (one in the intensive care unit), with no deaths. Even in a middle-income country, COVID-19 was reported to be relatively mild in sickle cell patients. In relation to risk factors, blood type O seems to confer some protection against developing severe COVID-19, a finding that could guide clinicians to adopt more clinical surveillance for patients with non-O blood type in sickle cell patients.

Long-Term Effects of Allogeneic Hematopoietic Stem Cell Transplantation on Systemic Inflammation in Sickle Cell Disease Patients.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only currently available curative treatment for sickle cell disease (SCD). However, the effects of HSCT on SCD pathophysiology are poorly elucidated. Here, we assessed red blood cell (RBC) adhesiveness, intensity of hemolysis, vascular tone markers and systemic inflammation, in SCD patients treated with allogeneic HSCT. Thirty-two SCD patients were evaluated before and on long-term follow-up after HSCT. Overall survival was 94% with no severe (grade III-IV) graft-vs-host disease and a 22% rejection rate (graft failure). Hematological parameters, reticulocyte counts, and levels of lactate dehydrogenase (LDH), endothelin-1 and VCAM-1 normalized in SCD patients post-HSCT. Expression of adhesion molecules on reticulocytes and RBC was lower in patients with sustained engraftment. Levels of IL-18, IL-15 and LDH were higher in patients that developed graft failure. Increased levels of plasma pro-inflammatory cytokines, mainly TNF-α, were found in SCD patients long-term after transplantation. SCD patients with sustained engraftment after allo-HSCT showed decreased reticulocyte counts and adhesiveness, diminished hemolysis, and lower levels of vascular tonus markers. Nevertheless, systemic inflammation persists for at least five years after transplantation, indicating that allo-HSCT does not equally affect all aspects of SCD pathophysiology.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. V: Manufacture and quality control.

Chimeric antigen receptor T cells (CAR-T), especially against CD19 marker, present in lymphomas and acute B leukemia, enabled a revolution in the treatment of hematologic neoplastic diseases. The manufacture of CAR-T cells requires the adoption of GMP-compatible methods and it demands the collection of mononuclear cells from the patient (or from the donor), generally through the apheresis procedure, T cell selection, activation, transduction and expansion ex vivo, and finally storage, usually cryopreserved, until the moment of their use. An important aspect is the quality control testing of the final product, for example, the characterization of its identity and purity, tests to detect any contamination by microorganisms (bacteria, fungi, and mycoplasma) and its potency. The product thawing and intravenous infusion do not differ much from what is established for the hematopoietic progenitor cell product. After infusion, it is important to check for the presence and concentration of CAR-T cells in the patient's peripheral blood, as well as to monitor their clinical impact, for instance, the occurrence of short-term, such as cytokine release syndrome and neurological complications, and long-term complications, which require patient follow-up for many years.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VI: Accreditation process.

The adherence to accreditation programs proves the institutions' voluntary effort to pursue the quality and safety of their products and services by meeting internationally accepted standards audited by experts in the field, external to the service. Meeting such standards often exceeds domestic legal requirements. However, service providers are not released from complying with the legal requirements, both local and international, pertinent to the field. Accreditation programs use the precepts of the quality management system to validate and standardize processes, monitor results through quality control, proficiency testing, and indicators, and perform risk management. For cellular therapy services, the assessing agencies available in our field are the AABB/ABHH (American Association of Blood Banks/Brazilian Association of Hematology, Hemotherapy and Cellular Therapy) and FACT-JACIE (Foundation for the Accreditation of Cellular Therapy-Joint Accreditation Committee, ISCT/EBMT). Both agencies require that the accredited organization meets all the standards defined in each program. Applying services also have to establish and comply with a quality management standard that demonstrates procedural interrelationship to ensure product and service quality. This paper aims to concisely outline the essential features of those two accreditation programs, along with a brief overview of the accreditation process under each of them.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VII. Present and future of technologies for production of CAR cell therapies.

Chimeric Antigen Receptor T (CAR-T) cells are certainly an important therapy for patients with relapsed and/or refractory hematologic malignancies. Currently, there are five CAR-T cell products approved by the FDA but several research groups and/or biopharmaceutical companies are encouraged to develop new products based on CAR cells using T or other cell types. Production of CAR cells requires intensive work from the basic, pre-clinical to translational levels, aiming to overcome technical difficulties and failure in the production. At least five key common steps are needed for the manipulation of T-lymphocytes (or other cells), such as: cell type selection, activation, gene delivery, cell expansion and final product formulation. However, reproducible manufacturing of high-quality clinical-grade CAR cell products is still required to apply this technology to a greater number of patients. This chapter will discuss the present and future development of new CAR designs that are safer and more effective to improve this therapy, achieving more selective killing of malignant cells and less toxicity to be applied in the clinical setting.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. Special article: compassionate use and clinical trial on CAR-T cells.

There are only two ways for a patient to gain access to treatment with an experimental product, such as CAR-T cells: participate in a clinical trial or receive a product in a compassionate basis. In the first case, the main beneficiary is society itself, which may in turn obtain a new treatment paradigm for a specific disease. In the second case, the use of a medicinal product has the objective of care in benefit of patients in grave clinical condition, for which no approved medicinal products exist, or for which all the possibilities for benefit from standard therapies have been exhausted. The CAR-T cell therapy may be included in one or the other types of access. The compassionate use is not a specific type of clinical research and should therefore not have its use appreciated by a research ethics committee, but rather by the medical ethics committee at the institution where the treatment will take place and by the regulatory agency.