Introduction. Bioluminescent labeling of tumor cells is becoming the standard in preclinical studies of novel anti-cancer drugs. Despite the variety of luciferases, many are not suitable for in vivo imaging.

Aim: to compare different Nanolux substrates in vitro and demonstrate the feasibility of using the NLuc/h-coelenterazine pair for in vivo imaging of disseminated tumor cells using the IVIS Spectrum system.

Materials and methods. The target cell line and T-cells expressing chimeric antigen receptors (CAR) were obtained via lentiviral cell transduction. Luminescence was measured with a Luminoskan™ Microplate Luminometer, while in vivo imaging in an NSG mouse model of CAR T-cell therapy was performed using the IVIS Spectrum system.

Results. Various substrates were compared in vitro for luminescence brightness and stability.

Additionally, the use of h-coelenterazine in combination with Nluc luciferase was demonstrated for in vivo cell imaging in mice. A genetically modified Nalm6-NLuc-CopGFP cell line was successfully obtained.

Conclusion. The comparison of various Nluc substrates in vitro and in vivo identified an optimal enzyme/substrate pair, which can serve as a valuable tool for assessing efficacy, safety and toxicity of compounds and cellular products being developed for antitumor therapy. The plasmid encoding Nluc can also be used to modify other cell lines necessary for the development and characterization of new gene therapy approaches.

Introduction. An important characteristic of therapy with modified T-cells expressing the chimeric antigen receptor (CAR) is the duration of CAR T-cell persistence in the patient’s body. Prolonged re-stimulation of CAR T-cell product in vitro with further analysis of its subpopulation composition and cytotoxic activity is one of the approaches to model cell behavior in patients.

Aim: to study the immunophenotype, exhaustion and functional characteristics of anti-CD19 CAR T-cell products derived from healthy donors and patients with B-cell acute lymphoblastic leukemia (B-ALL) at prolonged re-stimulation conditions in vitro.

Materials and methods. Anti-CD19 CAR T-cell products were generated from 5 healthy donors and 3 patients with B-ALL. The immunophenotype of the obtained cell products was studied under conditions of repeated antigenic re-stimulation in vitro using the target cell line NALM6 carrying the CD19 antigen over 7-10 days. At each experimental timepoint, the following parameters were assessed: proliferative and cytotoxic activity, expression of exhaustion markers, and changes in the subpopulation composition of CAR T-memory cells (immunophenotype).

Results. Anti-CD19 CAR T-cells had significant cytotoxic activity regardless of the lymphocyte source (healthy donors/patients). During prolonged antigenic re-stimulation of all products, a decrease in the proportion of naive (TN) and effector (TE) CAR T-cells, and an increase in the proportion of central (TCM) and effector memory (TEM) cells was observed. CAR T-cells showed increased expression of exhaustion markers (PD1, TIM3) irrespective of the origin of the cell and E:T ratio.

Conclusion. Analysis of cytotoxic activity and immunophenotypic composition of CAR T-cell products under conditions of prolonged re-stimulation revealed a trend toward decreased cytotoxicity, as well as differences in proliferation dynamics and population composition between cell products obtained from B-ALL patients and healthy donors.

Introduction. A multidisciplinary emergency hospital is a significant consumer of platelet concentrates (PCs). The specificity of requirements, logistics, and clinical use of PCs in multidisciplinary hospitals may affect the strategy for procuring this blood component.

Aim: to analyze the practice of PC transfusion in a multidisciplinary hospital providing emergency and urgent care to the population.

Materials and methods. In a retrospective observational study spanning over a 5-year period, 7,049 PC transfusions were examined in an emergency care multidisciplinary hospital. The study analyzed the indications for PC transfusion, PC efficacy, transfusion load on the recipient, urgency of performing PC transfusion, group compatibility of used PCs, PC type, and compared the corrected count increment (CCI) 24 hours after transfusion of various PC types.

Results. Between 23 % and 29 % of all PCs were transfused to patients who were admitted with polytrauma and massive blood loss, 18–27% were transfused to patients with septic complications, 8–13 % to patients during preparation and postoperative management for liver transplantation, 21 % to patients who underwent ECMO. Emergency indications accounted for 31.8–40.7% of PC transfusions. In 61.5 % of cases, the indication was hemorrhagic syndrome in combination with thrombocytopenia. AB0-identical transfusions were performed in 4,723 (67.1 %) cases. There were no significant differences in CCI when using apheresis, pooled or cryopreserved PCs.

Conclusion. When planning the work of hospitals providing emergency medical care to patients with surgical and traumatological profiles, the need for PC transfusions in 1.5–2 % of patients should be considered. Emergency supply can be facilitated using the “platelet management” tools: a reserve of universal donor PCs-prepared in additive solution or obtained apheresis from AB-group donors or via pooling from O-group donorsand cryopreserved PCs.

Introduction. The development of thrombotic microangiopathy of critical illnesses (TTP-like syndrome) is traditionally considered from the standpoint of imbalance in the von Willebrand factor system — ADAMTS-13, while coagulation disorders remain poorly studied.

Aim: to study the dynamics of fibrin clot formation and thrombin generation in patients with TTP-like syndrome, as well as their relationship with the severity of endothelial activation/damage, the activity of natural anticoagulant systems and complement systems.

Materials and methods. A prospective observational cohort study included 76 patients who underwent surgical treatment for heart disease and developed TTP-like syndrome as a postoperative complication. Inclusion criteria: multiple organ failure, thrombocytopenia <100×10⁹/l three days after surgery, schistocytosis >1%. The dynamics of fibrin formation and thrombin generation were assessed using the Thrombodynamics test; concentrations of natural anticoagulants, markers of endothelial damage, and the activity of complement system components were also evaluated.

Results. The mortality rate was 40.8% (31 patients). The analysis of three models revealed: 1) the complement system determines thrombin generation; 2) an increase in the concentration of natural anticoagulants, primarily thrombomodulin, inhibits the generation of thrombin on the activator and the propagation of its activation wave; 3) the degree of endothelial damage, starting with desquamation of the glycocalyx (reflected in an increase in plasma concentrations of syndecan-1 and heparan sulfate) and ending with the destruction of intercellular contacts and necrosis of endothelial cells with the release of PECAM и VE-cadherin; 4) endotheliopathy leads to platelet activation and consumption, as well as tissue hypoxia.

Conclusion. TTP-like syndrome is characterized by inhibition of thrombin generation, the degree of which depends on the severity of endothelial damage. This is due to the activation of natural anticoagulant systems. Desquamation of the endothelial glycocalyx reduces its resistance to the terminal complement complex which increases endothelial damage. In parallel, complement-mediated activation of platelets occurs. Deep endothelial damage, as well as the development of arterial microthrombosis, despite low thrombin generation, lead to an increase in hypoxic organ damage and poorer treatment outcomes.

Introduction. Endoprosthetics remains the only method of treating end-stage arthropathy in patients with hemophilia. Along with the increase in the number of endoprosthetics, the number of periprosthetic infections is also on the rise.

Objective. to determine the most common pathogens and their sensitivity to antimicrobial drugs, as well as risk factors for the development of periprosthetic infections in patients with hemophilia after endoprosthetics of large joints.

Materials and methods. A retrospective study analyzed cases of PJI of large joints in patients with hemophilia between 2015 and 2022, along with the causative agents of this complication.

Results. A total of 80 cases of PJI were identified. In patients with hemophilia A, PJI occurred in 76.0% of cases (61 patients), in patients with hemophilia B in 24.0% of cases (19 patients). Primary infection was detected in 47.5% of cases (38 PJI). Relapse of infection was detected in 42 episodes (52.5%). A washing system with dioxidine or polyhexadine was used in 9 cases (11.2%) of primary infection, in all other cases a two-stage revision tactic was used. 20 different pathogens were identified: 58 (65.0%) episodes of gram-positive flora, 13 (14.0%) cases of gram-negative flora, 3 (3.0%) episodes of fungal infection. It was not possible to identify the pathogen in 16 (18.0%) cases. The most common pathogens were S. aureus (21.1%) and S. epidermidis (21.1%), E. faecalis (7.3%). Among Staphylococcus aureus isolates, 18.8% were methicillin-resistant strains. Among gram-negative bacteria, P. aeruginosa was detected in 6 cases (7.3%), all of which exhibited multiple antibiotic resistance. The number of cases of PJI that arose in the first month or first year did not differ significantly from episodes when infectious complications arose in the period of a year or more. PJI after revision endoprosthetics most often debuted within 12 months after surgery.

Conclusion. The highest risk of PJI in patients with hemophilia occurs within the first year after revision endoprosthetics. PJI develops more frequently in patients with hemophilia than the general population, which is attributed to a greater number of risk factors in patients with hemophilia compared to the general patient population.

Introduction: Glucose-6-phosphate isomerase (GPI) deficiency is a hereditary nonspherocytic hemolytic anemia with an autosomal recessive inheritance pattern.

Aim: to study the clinical and genetic data of patients with GPI deficiency.

Patients and methods: A single-center retrospective study analyzed medical records of patients under 18 years of age who were admitted to the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology in the period between 2018 to 2024. The analysis included 13 patients from 12 families, 2 girls and 11 boys, with a median age at diagnosis of 3.5 years.

Results: All patients had hemolytic anemia of different verity and 2 patients from the same family had neurological symptoms (epilepsy, tetraparesis, psychomotor retardation). Three patients underwent splenectomy due to high transfusion dependence, after which they showed improvement, but hemolysis was not completely stopped. A total of 14 different mutations in the GPI gene were identified, 8 of which had not been previously described. Their pathogenicity was determined using various algorithms.

Conclusion: GPI deficiency is characterized by high clinical variability, requiring an individualized approach to diagnosis and therapy. The obtained results confirm the importance of a comprehensive examination, including laboratory and genetic testing.

Introduction. The production cycle of CAR T-cell product includes several sequential stages, each of which may infl uence the efficiency of transgenic cells: obtaining the patient’s cellular material, isolating the target T-lymphocyte population, activation, transduction of the cells with a viral vector carrying the CAR construct, and expansion of the obtained CAR T-cells with further administration to the patient.

Aim: to review the impact of each of the production steps on the cell product performance in both in vitro and in vivo experiments, as well as clinical applications.

Main findings. The manufacturing characteristics of various CAR T-cell products were analyzed in this review, followed by a discussion on how different manufacturing characteristics and chimeric antigen receptor structures affect the antitumor efficacy and safety profi le of the cell product. The production of a CAR T-cell product is a multifactorial process that requires optimization of parameters and must take into account the characteristics of the initial raw material (T-lymphocytes).

Introduction. The introduction of chimeric antigen receptor (CAR) T-cell therapy into clinical practice has transformed international treatment standards for B-cell leukemias, lymphomas, and multiple myeloma. Alongside its high antitumor activity, CAR T-cell therapy is associated with a unique profile of adverse events, including cytokine release syndrome (CRS), immune cell-associated neurotoxic syndrome (ICANS), and macrophage activation syndrome (MAS).

Aim: To present data on the mechanisms of immune toxicity of CAR T-cell therapy, its clinical manifestations, as well as prevention and treatment strategies.

Basic information. CRS is a self-sustaining hyperinflammatory condition with a specific spectrum of clinical manifestations, driven by endothelial activation and increased permeability. Genetic predisposition to CRS is associated with polymorphisms in genes involved in immune cell adhesion and activation. Key stages of pathogenesis include hyperproduction of cytokines, particularly interleukins (IL)-1β, 6, 8, 10, and interferon-γ, reduced expression of endothelial adhesion molecules, overproduction of permeability factors, and consequent interstitial organ edema and dysfunction. Conditions closely associated with CRS include immune effector cell-associated neurotoxicity syndrome (ICANS) and macrophage activation syndrome/secondary hemophagocytic lymphohistiocytosis (MAS). Treatment of CRS is based on the use of glucocorticosteroids and anticytokine monoclonal antibodies targeting the IL-6 receptor, IL-6 itself, IL-1, and interferon-γ. However, a significant proportion of adverse outcomes are driven by ICANS and MAS. The most promising treatment approach for these conditions currently involves the use of interleukin-1 antagonists, which may mitigate these severe immune toxicities.

Introduction. Traditional methods of pre-transplant conditioning provoke acute organ damage. Immunotherapeutic drugs that target various markers of blood cells are safer. This potential is possessed by drugs based on cytotoxic immune cells. Their use may concurrently address two treatment objectives — residual tumor clearance (induction of remission) and myeloablation — consequently improving graft acceptance while preventing graft-versus-host disease development.

Aim: to systematize information on the use of cell therapy in patients with hematologic malignancies and to evaluate the role of such therapy in preparing patients for hematopoietic stem cell transplantation (HSCT).

Main findings. Current conditioning regimens and their rationale were reviewed. Approaches to the use of immunotherapeutic agents based on cytotoxic immune cells for the treatment of patients with hematologic neoplasms are discussed, and the role of such therapy in preparing patients for HSCT is outlined.

Introduction. Follicular lymphoma (FL) is the most common indolent lymphoma. Despite the long overall survival, the tumor remains incurable in most patients. Currently, there is no specific algorithm for choosing therapy for ≥3 lines of patients with FL. CAR T-cell therapy (therapy with genetically modified T-lymphocytes carrying a chimeric antigen receptor — CAR) has proven effective in many tumors, including FL.

Aim. to systematize data on the efficacy and safety of modern CAR T-cell therapy for follicular lymphoma.

Main findings. CAR T-cell therapy is an effective option both for patients with FL after multiple courses of therapy, and for refractory tumors. Ongoing advancements continue to refine this method, enhancing its safety and expanding the possibilities of use. CAR T-cell therapy is increasingly being integrated into anti-relapse regimens for FL.

Introduction. The highly immunogenic D antigen is one of the most important antigens after the group antigens A and B. Numerous alleles of the RHD gene lead to the emergence of new D antigen phenotypes. The Del phenotype is characterized by an extremely weak expression of the D antigen.

Aim: to present the results of D antigen typing in a blood donor with the Del phenotype.

Materials and methods. Two technologies were used for serologic typing of donor blood: microcolumn agglutination technology and solid phase microplate Capture technology. Primary genotyping of the donor was performed by allele-specific polymerase chain reaction (ASP-PCR). Nucleotide substitutions were searched for by Sanger sequencing of PCR products of exons of the RHD gene.

Results. When serological typing with IgM and IgG anti-D antibodies using solid-phase microplate technology was performed, a positive result was obtained. When determining partial variants of the D antigen, it was not possible to unambiguously interpret the data obtained. Molecular genetic analysis was required to identify variants of the weak D antigen. The results of primary genotyping by ASP-PCR suggested deletion of exon 9 of the RHD gene or nucleotide substitution in exon 9. According to the results of Sanger sequencing, a variant of the DNA nucleotide sequence c.1203T>A was detected in exon 9, resulting in the formation of the stop codon Tyr401Ter (rs759513820). This genetic variant corresponds to the RHD*01EL.17 allele. The antigen encoded by this allele is of the Del type.

Conclusion. It is necessary to use immunohematological methods in combination with molecular genetic methods for D antigen typing in blood donors with the Del phenotype in order to prevent alloimmunization with the D antigen.