The characterization and frequency of diagnosis of EBV-positive variants with a lymphoid predominance in the structure of Hodgkin lymphoma

Introduction. Epstein — Barr virus (EBV) plays an important role in the pathogenesis of lymphoid tumors, in particular Hodgkin lymphoma. The frequency of expression of the EBV varies in different histological variants of classical Hodgkin lymphoma and is rarely observed in nodular lymphocyte predominant Hodgkin lymphoma.Aim — to study the pathomorphological features of the histological variants of Hodgkin lymphoma with lymphoid predominance associated with the EBV, as well as the frequency of their diagnosis in the structure of Hodgkin lymphoma.Materials and methods. The retrospective study included 794 patients with a verified diagnosis of Hodgkin lymphoma using histological and immunohistochemical methods on biopsy material for the period 2018–2019 (age range — 18–91 years old; median — 34 years old; men : women — 1.1 : 1). The presence of EBV in biopsies was assessed by immunohistochemical reaction with antibodies to EBV (clone LMP1), or by chromogenic in situ hybridization with probes for EBV-encoded small RNAs.Results. Classical Hodgkin lymphoma was diagnosed in 91 % (725/794) cases, nodular lymphocyte predominant Hodgkin lymphoma — in 9 % (69/794) cases. EBV-positive Hodgkin lymphoma accounted for 11 % (82/725) of all cases of classical Hodgkin lymphoma, (age range — 18–81 years old, median — 45 years old; men : women — 2.5 : 1). All cases of nodular lymphocyte predominant Hodgkin lymphoma were EBV-negative. Lymphocyte-rich classical Hodgkin lymphoma was found in 14 patients (14/725, 2 %), 4 patients showed intermediate morphoimmunohistochemical features with nodular lymphocyte predominant Hodgkin lymphoma, which were statistically significantly different from classical Hodgkin lymphoma by the presence of B-zones in the form of large nodules (p = 0.0157) and expression CD20 by tumor cells (p = 0.0404).Conclusion. Nodular lymphocyte predominant Hodgkin lymphoma is not characterized by a connection with EBV infection, unlike classical variant — lymphocyte-rich classical Hodgkin lymphoma. The obtained data support the concept of the existence of a transient form of Hodgkin lymphoma, which has the features of nodular lymphocyte predominant Hodgkin lymphoma and classical Hodgkin lymphoma, in the pathogenesis of which the Epstein — Barr virus likely plays a role.

Prognostic value of minimal residual disease before allogeneic hematopoietic stem cell transplantation in patients with acute leukemia

Introduction. One of the main causes of treatment failure after allogeneic hematopoietic stem cells transplantation (alloHSCT) for acute leukemia (AL) is disease relapse. In recent years, multiparameter fl ow cytometry (MPC) has been widely used to detect minimal residual disease (MRD) because of its capacity to identify patients with a high risk of relapse due to availability and the ability to obtain results in a timely manner.Aim — to evaluate the prognostic value of MRD status before allo-HSCT and the effect of donor type and conditioning intensity on long-term results of allo-HSCT of MOB-positive patients.Patients and methods. The analysis included 107 patients with acute myeloid leukemia (AML) and 63 patients with acute lymphoblastic leukemia (ALL) who underwent allo-HSCT between September 2015 and June 2020. All patients were in complete morphological remission before allo-HSCT. At the time of allo-HSCT 91 patients with AML and 37 patients with ALL were in the first complete remission (CR), in their second and more than two CRs were 16 and 26 patients, respectively. The median follow-up was 18 (1.5–48) months for AML and 14 (1.8–60.1) months for ALL. Immunophenotypic study was performed before allo-HSCT. MRD was detected using a combination of the “different from normal” method and the search for cells with a leukemia-associated immunophenotype.Results. The disease status at the time of transplantation and the presence of MRD before allo-HSCT were independent factors infl uencing the probability of relapse (disease status: HR = 2.911, 95% CI: 1.328–6.379; MRD before allo-HSCT: HR = 7.667, 95% CI: 3.606–16.304) and post-transplant mortality (disease status: HR = 2.911, 95% CI: 1.328–6.379; MRD before allo-HSCT: HR = 7.667, 95% CI: 3.606–16.304). In univariate analysis, the relapse-free survival of MRD+ patients with AL in the first CR was significantly worse than in MRD– (AML: 23 % versus 57 %, p < 0.0001, ALL: 34 % versus 61.7 %, p = 0.0484), and the probability of relapse in MRD+ patients was significantly higher (AML: 75 % versus 12 %, p < 0.0001, ALL: 57 % versus 7 %, p = 0.0072). Pre-transplant MRD status was not prognostically significant for AL-patients in the second and third remission. The development of chronic GVHD reduces post-transplant mortality if it does not require systemic therapy with glucocorticosteroids (HR = 0.006, 95% CI: 0.008–0.446).Conclusion. Testing for MRD of patients with AL in the first CR before allo-HSCT is necessary for risk stratification and identification of patients who will need preventive post-transplant therapy in order to prevent disease relapse.

Thermolability of factor VIII in donor fresh frozen blood plasma

A criterion of the quality of fresh frozen blood plasma (FFP) is the activity of clotting factor VIII (FVIII).Aim — to identify technological barriers in the study of FVIII thermolability and to describe the requirements for experiments, providing new knowledge about the thermolability of this factor.Basic information. An analysis of domestic and foreign publications devoted to the study of the mechanisms responsible for reducing the value of FVIII activity in donor blood plasma from the moment of donation to the moment of transfusion was carried out. Data on the decrease in FVIII activity at various stages of work with blood plasma are presented. An analysis of methods for preparing samples for studying changes in the values of FVIII in donor blood plasma was performed. The existence of contradictory conclusions about the infl uence on the change in FVIII at the thawing stage of various values of the effects on FFP and poor knowledge of the change in the indicator at the stage of heating to the transfusion temperature after the end of the phase transition in the samples was established. The fundamental differences in the methods of preparing and conducting experiments in previous works are determined. Methods for increasing the reliability of experimental results for studying the thermal lability of FVIII are proposed.

Early and late relapses of multiple myeloma after autologous haematopoietic stem cell transplantation

Introduction. Autologous haematopoietic stem cell transplantation (auto-HSCT) is a highly effective treatment for multiple myeloma (MM). Auto-HSCT allows a signifi cant improvement of haematological response leading to higher overall survival and quality of life in MM patients. Nonetheless, the majority of patients develop relapse.Aim — a comparison of clinical MM relapses developing at variant terms after auto-HSCT.Patients and methods. A retrospective study enrolled 65 MM patients aged between 39 and 64 years. All patients had auto-HSCT during 2009–2019, all had achieved complete response (CR) or very good partial response (VGPR) and all since developed immunochemical MM relapse in laboratory evidence. Patients were divided in two cohorts by relapse term, the early (within 12 months of auto-HSCT) and late relapse.Results. Early immunochemical relapse was diagnosed in 13 (20 %), late relapse — in 52 (80 %) patients. The dependence between relapse term and depth of post-auto-HSCT antitumour response has been determined. The proportion of CR patients was signifi cantly higher in late than in early relapse (55.8 vs. 23 %). In follow-up, 60 patients (92.3 %) were initiated on antirelapse therapy, all early relapse and 90.3 % late relapse patients. On day +100 of auto-HSCT, CR patients had later relapse vs. VGPR individuals (median 24 vs. 19.9 months, p = 0.08) with signifi cantly weaker paraprotein secretion resembling the clinical course of monoclonal gammopathy of unclear signifi cance (MGUS).Conclusion. Auto-HSCT allows long-term control of the disease. A signifi cant prognostic factor is antitumour response on +100 day of auto-HSCT. Patients attaining CR have later relapse progressing in a MGUS-like manner. Patients with late indolent relapse can be managed long-term without antitumour therapy.

Comparative study of the effect of tranexamic acid and exogenous fibrin monomer on fibrin formation in the injury area during stimulation of fibrinolysis by streptokinase

Introduction. Earlier studies of low-dose fibrin monomer (FM) demonstrated that low-dose FM has unique hemostatic properties in vivo.Aim — to compare the morphological consequences of intravenous administration of tranexamic acid (TXA) and FM with the hemostatic and hemostasiological effects in hypofibrinogenemia caused by the use of streptokinase after controlled liver injury.Materials and methods. The morphological pattern of fibrin formation in the liver injury area after spontaneous arrest of bleeding in the animals treated with streptokinase or placebo was studied in 73 male rabbits of the Chinchilla breed, split into four groups. In three groups, the study was performed under the conditions of intravenous administration of placebo, TXA, or FM against the background of fibrinolysis activation by streptokinase. Platelet count in the blood, the concentration of fibrinogen, as well as the results of calibrated thrombography, were taken into account.Results. Sequential administration of streptokinase and TXA was accompanied by decreased fibrinogen concentration (by 29.6 %) and, at the same time, a reduction in blood loss (by 15.4 times) in comparison with animals where placebo was used instead of TXA. A decrease in blood loss was associated with increased thickness of thrombotic deposits at the edge of the wound, mainly consisting of red blood cells. These observations were combined with data on the acceleration of thrombin formation in venous blood plasma in a calibrated thrombography test (Peak thrombin 65.4 nmol/L to 109.6 nmol/L in the placebo group). Compared to the observations where placebo was administered instead of FM, however, the sequential use of streptokinase and FM also led to a decrease in blood loss (by 11.0 times) despite decreased fibrinogen concentration (by 23.3 %). A decrease in blood loss was also associated with platelet consumption in venous blood and with increased thickness of thrombotic deposits on the injury surface, where, in addition to red blood cells, the accumulation of fibrin masses was determined by the morphological pattern.Conclusion. The mechanisms of the systemic hemostatic effect of TXA and FM are different, despite the similarity of the achieved hemostatic effects in the conditions of stimulation of blood fibrinolytic activity. These findings expand the understanding of new therapeutic possibilities for reducing post-traumatic blood loss.

Application of conditioned medium from mesenchymal stromal cells in the protocol for ex vivo production of megakaryocytes and platelets

Introduction. Of interest is the use of a conditioned medium from mesenchymal stromal cells in order to increase the expansion of CD34+ hematopoietic stem cells (HSCs).Aim — to analyze the effi cacy of two methods of ex vivo production of human megakaryocytes and platelets from CD34+ cord blood HSC using conditioned media from mesenchymal stromal cells and IMDM. Methods. Two cultivation methods that differ from each other by medium composition were compared. As a control of antigen expression of the donor, venous blood platelets were used. CD34+ HSCs were isolated from mononuclear fraction of cord blood using the immunomagnetic selection technique. The resulting cells were introduced at a concentration of 1 × 104 cells/mL into 24-well plates and cultured at 39 °C and 10 % CO2 for the first 7 days, after which the conditions were changed to 37 °C and 5 % CO2 and cultured for 14 days. In Group 1, up to day 7, the culture was performed using conditioned medium from mesenchymal stromal cell containing TPO (30 ng/mL), SCF (2 ng/mL), IL-6 (7.5 ng/mL), IL-9 (13.5 ng/mL), and in Group 2 a IMDM medium with the same cytokine cocktail was used. The cells were calculated using haemocytometer. CD34, CD41a, CD42b expression was evaluated using fl ow cytometry. Statistic data was processed with using R-language. The differences were evaluated as statistically signifi cant at signifi cance level p < 0.05.Results. Megakaryocyte production was observed starting from day 7 of culture. The expression level using conditioned medium from mesenchymal stromal cells (Group 1) according to CD41a was 5.84 ± 0.33 % versus 10.43 ± 1.08 % using IMDM medium (Group 2). On day 13 the ratio increased up to 42.05 ± 1.71 % in Group 1 and 61.78 ± 1.71 % in Group 2. CD41a+ megakaryocytes of Group 1 expressed the CD42b marker at the level of 96.85 ± 1.06 % versus 88.7 ± 0.56 % in Group 2. With the application of MSC conditioned medium the average number of nucleated cells was signifi cantly higher on the day 11 and it was equal 326.016 ± 1.86 × 104 cells/mL vs 197.26 ± 10.55 × 104 cells/mL in IMDM medium. Proplatelet formation was observed with microscopy staring from the day 12. The ratio of CD41a+ /CD42b+ platelets was 59.5 ± 3.85 % in conditioned medium, 65.9 ± 8.72 % in IMDM, and 96.11 ± 0.89 % in control platelets derived from venous blood.Conclusion. It was demonstrated that the use of MSC conditioned medium leads to an increase in the expansion of nucleated cells, however it decreases the rate of differentiation in megakaryocytes.

Regional hematology service registration system for the Russian Federation

Introduction. The hematology service registration system describes actual indicators of hematology service infrastructure and target objects of both assigned and affiliated organizations of Russian Federation regions along with its correlations. This system data base makes it possible to collect, save, and analyze information, creating a data storage library about all current existing resources and taking into account concrete, specific conditions of specialized and high-tech hematological medical assistance organizations at the local levels and formed quality and availability optimization proposals.Aim — description and justification of the need to create a unified, complex, available to all users, informational reference system with the possibility of up-to-date maintenance and accounting that allows for the keep and control of the regional characteristics and hematology service indicators of the Russian Federation.General findings. The creation of and development of the hematology service registration system began in 2018, and work on the project continues. Four main phases were selected as the key stages of the hematology service pasportisation: analyses of the current data, verification of information, creation of a user-friendly tool, system updating. Objects to monitor: medical key and related institutions, universities, affiliated organizations, laboratory service. The data working stages: data collection, manual updating, package uploading. The information sources: official websites, data high-tech medical treatment web portal, web portals of the public health authorities, Compulsory Medical Insurance Fund website, The Unified Data Health Care System, license register of the Federal Service for Healthcare Surveillance, data of the Federal State Statistics Service, official documents of Ministry of Public Health. Based on the developed checklists and forms for entering additional information for specialists of the subjects of the Russian Federation, a reference book of the Ministry of Defense (Excel file format) was created. However, in the course of further remote interaction and conducting field events in the regions of the Russian Federation during 2019–2020 new data needs for evaluating hematology services were determined. The increase of additional data inputs and its structure complication became the main reasons for further developing a new platform design concept and data conversion in database framework of the hematology service registration system (Access file format). The current objectives are as follows: authentication of the internal objects identifiers, implementation of the relational database codifier, modification of cartographic display and objects routing. The hematology service registration system contains data both for the key infrastructure nodes of Russian Federation hematology service and correlations within routing of all key system objects. Based on the positive results, the hematology service registration system is planned to be used by experts of specialized medical organizations, regional executive authorities on public health and professional communities, who will be able to accumulate all changes and indicators in dynamics within one system. The expected result is to provide the Ministry of Defense of the Russian Federation regions with methodological and informational support in specialized and high-tech medical care administration.

Essential thrombocythemia in children and adolescents — analysis of 31 cases

Introduction. Essential thrombocythemia is an extremely rare disorder in childhood. This disease is characterized by a persistent increase in the peripheral blood platelet count, associated with a proliferation of atypical megakaryocytes in the bone marrow.Aim — to analyze the clinical features of the course of essential thrombocythemia (ET) and the response to therapy in pediatric and adolescent patientsMaterials and methods. Thirty-one patients with ET under the age of 21 years were analyzed. All patients were diagnosed with ET in accordance with WHO criteria on the basis of an examination, including assessment of clinical data, laboratory tests (general clinical tests; morphological, genetic, and histological examinations of bone marrow), instrumental studies, and an assessment of response to treatment.Results. The average age of disease onset was 9 years 9 months, with a median of 9 years 6 months. Organomegaly was recorded in 16 (52 %) patients, of whom 6 (37.5 %) had isolated splenomegaly and 6 (37.5 %) had hepatosplenomegaly. Bleeding was noted in 6 (19.4 %) patients with a deep decrease in vWF:RCo (no more than 15 %) and an extreme increase in platelets (PLT) (more than 2000 × 109 /L). Twelve (38.7 %) patients suffered from microcirculation disorders (headaches, dizziness, melalgia), half of them had a platelet count of 1000–2000 × 109 /L, which is comparable to asymptomatic patients. No thrombosis was registered in our group. The JAK2V617F mutation was detected in 3 (9.7 %) patients, a mutation in the CALR gene was found in 9 (29.0 %) of patients, there was a mutation in the MPL gene in one (3.2 %) patient, and in the remaining cases (18 (58.1 %) patients), there was no damage to typical driver genes. Translocation t(12;12) was revealed in 1 (3.2 %) patient. The response to one-component cytoreductive therapy (CR+PR) was found to be quite high in young patients and constituted about 70–80 %. The complete response rate (CR) was as follows, respectively: 42.9 % (3) — to anagrelide therapy (ANA), 47.4 % (9) — to interferon therapy (INF), and 0 % — to hydroxycarbamide (HU). However, HU was not used in the fi rst line of therapy for the children in our group.Conclusion. In the pediatric population, ET patients are dominated by the group of “triple-negative” disease, which somewhat complicates the differential diagnosis with secondary thrombocytosis. Compared to the adult population, the risk of bleeding is higher for pediatric patients, which is associated with the large number of patients with extremely high levels of platelets. In the case of hemorrhagic syndrome development or microcirculatory disorders that cannot be stopped by taking antiplatelet agents, we recommend giving preference to INF and HU as fi rst-line therapy, due to the peculiarities of pharmacokinetics and the potential risk of progression of myelofi brosis during ANA therapy.

Expression of PD-1 and TIM-3 inhibitory checkpoint molecules by T-lymphocytes in early post-transplant period in multiple myeloma patients

Introduction. High-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (auto-HSCT) is the standard of treatment for multiple myeloma (MM) patients. The post-transplant period appears to be promising for targeted anti-checkpoint therapy in MM.Aim — to study the dynamics and functional properties of T-cells expressing inhibitory checkpoint molecules PD-1 and TIM-3 in patients with MM under conditions of lymphopenia after HDC with auto-HSCT.Methods. The study included 40 patients with MM who underwent HDC with auto-HSCT. The counts of PD-1- and TIM3-positive CD8+ and CD4+ T-cells and their functional activity on the intracellular expression of Ki-67, production of granzyme B, and interferon-γ were assessed by fl ow cytometry.Results. Relative counts of patient PD-1+ and TIM-3+ subsets of CD8+ and CD4+ T-cells obtained from bone marrow samples were higher compared to peripheral blood. CD8+ PD-1+ and CD4+ PD-1+ T-cells of MM patients had a pronounced cytotoxic and cytokine-producing potential. The functional activity of CD8+ TIM-3+ and CD4+ TIM-3+ T-cells was signifi cantly reduced compared with TIM-3-negative subsets. Low functional activity was also detected in populations of CD8+ and CD4+ T-lympho cytes, co-expressing PD-1 and TIM-3. The frequencies of T-cells expressing PD-1 and TIM-3 increased signifi cantly on the engraftment day after auto-HSCT. The proliferative activity of PD-1+ and TIM-3+ CD4+ and CD8+ T-cells and the cytotoxic potential of PD-1+ and TIM-3+ CD8+ T-cells were also signifi cantly increased compared to the data prior auto-HSCT.Conclusions. PD-1-positive T-cells in MM patients are related to activated or “early dysfunctional” but not exhausted subsets, while T-cells exhaustion is more analogous with CD8+ TIM-3+ and CD4+ TIM-3+ T-cells, as well as with subsets co-expressing PD-1 and TIM-3. To identify the state of T-cells exhaustion, it is necessary to evaluate T-cells subsets co-expressing PD-1, TIM-3, and other ICMs, and/or to study their functional properties. In the early post-transplant period, the proportion of Tcells expressing PD-1 and TIM-3 increases due to an increase in their proliferative potential.

Clonal hematopoiesis and its role in the development of hematological diseases

Introduction. The formation of blood cells in a healthy individual is ensured by polyclonal hematopoiesis. Recent studies have shown that with age, large clones with a common genetic marker are found in the peripheral blood, i. e. cells originating from a single progenitor cell. This phenomenon is called clonal hematopoiesis. In some cases, people with clonal hematopoiesis develop hematological diseases.Aim — to describe and summarize current data on the relationship between clonal hematopoiesis and hematological diseases.Main findings. This review describes the history of detection of clonal hematopoiesis, its main properties, the most frequent mutations in hematopoietic clones associated with the risk of transformation into myelodysplastic syndrome, and acute myeloid leukemia. The meaning and possible pathogenesis of tumor transformation are discussed.