Aplastic anemia (AA) is characterized by bone marrow (BM) aplasia and pancytopenia. BM stromal microenvironment is closely intertwined with hematopoietic cells by reciprocal regulation. It is still unclear how hematopoietic deficiency affects the bone marrow stroma of the AA patients. Multipotent mesenchymal stromal cells (MMSCs) are the progenitors of stromal cells. In vitro, proliferation rate of MMSCs of AA patients is decreased compared to those of healthy donors. This may be explained by the influence of pathological environmental condition in the patients’ BM. The aim of the study was to compare the effect of AA patients’ sera on healthy donor MMSCs to healthy donors’ sera and to elucidate the nature of their difference. Proliferation test showed 3-fold decrease in number of MMSCs after incubation in medium supplemented with AA patients’ sera compared to donors’ serum samples. The degree of this effect correlated with the severity of thrombocytopenia in patients. The decrease in cell number was not associated with cell death, as the number of apoptotic cells defined by flow cytometry did not differ between the groups. ELISA revealed a decreased level of PDGF-BB in the patients’ sera compared to donors’ serum samples (69 ± 5 pg/mL vs. 112 ± 21 pg/mL, respectively). The addition of recombinant PDGF-BB or healthy donor’s platelet lysate to the culture medium supplemented with AA patients’ serum restored its ability to support MMSCs growth. Thus, PDGF-BB deficiency is one of the environmental factors causing MMSCs damage in AA patients.

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Aplastic anemia (AA) is a rare disease manifested in hematopoiesis disruption due to decreased number and dysfunction of hematopoietic stem cells. According to severity of pancytopenia there are 3 forms of AA: non-severe AA (NAA), severe AA (SAA) and very severe AA. AA origin is mainly of autoimmune nature, but detailed pathogenesis is not fully described. The role of bone marrow (BM) stromal cells in the pathogenesis of AA is not clear. It is known that AA patients’ BM derived multipotent mesenchymal stromal cells (MMSCs) are altered in gene expression and proliferative and differentiation potential. However, the differences of MMSCs from patients with various forms of AA were not characterized due to the AA rarity and the difficulties in MMSCs cultivation. Characterization of BM stromal precursors in various forms of AA is important for understanding its pathogenesis. Aims: To reveal differences in proliferative properties, differentiation potential and gene expression pattern of NAA and TAA patients’ MMSCs compared to donors’ ones. Methods: The study included 22 patients with NAA (11m, 11f, 21-51 y.o., median 31) and 16 patients with TAA (8m, 8f, 18-63 y.o., median 27) at the onset of the disease. The control group included 30 healthy donors (17 m, 13 f, 14-61 y.o., median 28.5). Patients’ BM was obtained during a diagnostic punctures, donors one - during planned exfusions after informed consent was signed. MMSCs were isolated according to the standard method and cultured up to passage 3. Time to passage 0 (P0), the population doubling time (PDT), and the total cell production were determined. The differentiation potential of MMSCs was analyzed by expression level of marker genes for osteogenic (ALPL, PTHR1) and adipogenic (FABP4, PPARG) lineages after corresponding induction. MMSCs incubated in the standard medium were used as controls. Gene expression was analyzed with real-time PCR in the TaqMan modification. Data are presented as mean ± standard error of the mean. For normal distribution, the significance of differences was determined with Student t-test; otherwise, Mann-Whitney test was applied. Differences were considered significant at p<0.05. Results: Differentiation potential of MMSCs from patients with NAA, TAA, and donors did not differ between the groups. We revealed no differences in the total cell production of MMSCs in patients with NAA and TAA compared to donors, therefore, the proliferative potential of patients’ MMSCs is not altered (Table 1). The PDT of NAA patients’ MMSCs was increased compared to donors ones, which indicated a decrease in the proliferation rate of MMSCs in this group. The time to P0 in both groups of AA patients was longer than in donors. Gene expression had changed similarly in NAA and TAA MMSCs when compared to donors (Table 2). The expression of growth factor receptors genes (FGFR1, FGFR2, PDGFRA, PDGFRB) and NES was upregulated; the expression of some immune regulation (IL10, HLA-DRA) and regulation of hematopoiesis (ANGPT1) genes was downregulated. Upregulation of PDGFRA in NAA patients’ MMSCs compared to TAA patients and donors ones was found, probably, associated with a decrease in their proliferation rate.Summary/Conclusion: NAA, TAA patients’ MMSCs have a similar pattern of gene expression alterations; they do not differ from donors ones in proliferative and differentiation potential. NAA patients’ MMSCs reveal a decreased proliferation rate and upregulated PDGFRA gene expression. Keywords: Gene expression, Severe aplastic anemia, Aplastic anemia, Mesenchymal stem cell

The differentiation potential of individual clones of fibroblast CFU (CFU-F) was studied and the relative expression level of genes was analyzed in the culture of CFU-F from the bone marrow in patients with non-severe and severe forms of aplastic anemia at the onset of the disease. The differentiation potential of CFU-F clones was determined by the relative expression of marker genes using quantitative PCR. In aplastic anemia, the ratio of CFU-F clones with different differentiation potential changes, but the molecular mechanisms of this phenomenon are different in non-severe and severe aplastic anemia. In the culture of CFU-F in non-severe and severe aplastic anemia, the relative expression level of genes associated with the maintenance of the hematopoietic stem cell in the bone marrow niche changes, but the decrease in the expression of immunoregulatory genes occurs in severe form only, which may reflect differences in the pathogenesis of non-severe and severe aplastic anemia.

Background. Aplastic anemia proceeds with bone marrow failure and is associated with immunological suppression of normal blood stem cells’ proliferation, which lead to bone marrow aplasia. autoimmune aggression and internal defects of blood stem cell that cause abnormal hematopoiesis are being actively studied. An important role in the pathogenesis of the aplastic anemia is played by instability of telomere length (TL). determination of the initial TL makes it possible to clearly differentiate between the aplastic anemia and dyskeratosis congenita. also, it helps to identify the group of patients with short telomeres for prediction of therapy response. Aim. To investigation the TL of various blood and bone marrow cells in patients with aplastic anemia before treatment. Materials and methods. The group of patients with aplastic anemia was investigated (n = 45). blood donors (n = 32) and bone marrow donors (n = 10) of different ages were included in the reference group. adult patients with dyskeratosis congenita (n = 5) were included in the comparison group. Relative and absolute tl was identified in peripheral blood and bone marrow mononuclear cells, monocytes, lymphocytes by flow-FISH technique (combination of flow cytometry and fluorescence in situ hybridization). Results. Relative and absolute TL was comparable in different blood and bone marrow cells in patients with aplastic anemia before treatment. TL in peripheral blood and bone marrow mononuclear cells wasn’t significantly differed in groups of patients with aplastic anemia and healthy donors. Telomeres in patients with dyskeratosis congenita were identified as “ultrashort” and were significantly shorter than in patients with aplastic anemia. Conclusion. Determination of TL in patients with aplastic anemia is modern examination method, which is a necessary step of differential diagnosis between aplastic anemia and dyskeratosis congenita, which is the disease from group of constitutional bone marrow aplasia. It is preferred to identify the TL in adult patients with aplastic anemia by the flow-FISH. It is necessary to investigate the TL to predict treatment response and to identify risks of developing adverse experiences, which include relapse and clonal evolution.

The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are studied insufficiently. The aim of this work was to test the ability of MSC from patients with aplastic anemia to maintain hematopoietic precursors and to analyze the expression of genes associated with hematopoiesis and immune response. The ability of MSC to maintain hematopoietic precursors was determined by counting cobblestone area-forming cells; gene expression was analyzed by quantitative PCR. It was shown that MSC of patients with aplastic anemia preserve their ability to maintain hematopoietic precursors. Pronounced changes in the expression of the VEGFA and ANGPT1 genes were found. MSC from aplastic anemia patients with PNH clone significantly differ from those from aplastic anemia patients without PNH clone in terms of the expression of the SDF1, IL1R, and VEGFA genes. Changes in gene expression can be associated with the pathogenesis of the disease.

Introduction. The use of thrombopoietin receptor agonists, especially eltrombopag, in the treatment of aplastic anemia (AA) patients who did not respond to the previous immunosuppressive therapy (IST), is accompanied by the development of a hematological response in 40–60 % of patients.Aim — to study the effi cacy of using eltrombopag in treatment programs for AA patients refractory to previous IST.Methods. The study included 20 AA patients who were treated at the National Research Center for Hematology from 2015 to 2020. These patients did not respond to the conducted IST (ATG + CsA). Eltrombopag was administered at a dose of 150 mg/day. The results of treatment were assessed at 3 and 6 months: the achievement of hematological improvement, partial and complete remission, as well as the identifi cation of possible clonal evolution were determined.Results. Eleven out of 20 (55 %) patients responded to treatment: 2 patients developed hematological improvement, 6 patients — partial remission, 3 patients — complete remission. All 11 patients responded to treatment within 12 months from the start of eltrombopag, but further positive dynamics of hematological parameters are possible. The median duration of treatment with eltrombopag was 11 (1–48) months. Most of the patients were treated with eltrombopag in combination with CsA. The duration of the course of treatment with eltrombopag depended on the response received (stable hematological improvement, remission, as well as the detection of clonal evolution) or its absence and the need for ATG or BMT. An aberrant karyotype was found in 2 AA patients who received eltrombopag: in one patient monosomy of chromosome 7 was detected 1 month after the start of treatment, in another patient, 37 months later, a clone with a derivative of chromosome 16 from t(1;16) and subclone with complex disorders of the karyotype without signs of myelodysplasia in the bone marrow.Conclusion. The inclusion of the TPO receptor agonist eltrombopag in the treatment program for AA patients allows for a stable hematological response and remission of AA for patients who have not responded to IST. The effectiveness of eltrombopag is determined by adherence to the treatment algorithm, the optimal duration of the course, and the dose of the drug used. There is still a need for long-term observation of the patient and control morphological and cytogenetic studies.

Dyskeratosis congenita (DC) is a hereditary syndrome of bone marrow failure, which develops because of telomeres' defects and combines with cancer predisposition. Its classical clinical features are skin pigmentation, nail dystrophy, oral leukoplakia ("skin-mucosa triad"). The goal is to describe the algorithm of diagnosis, clinical specificities of DC and specific treatment for cases of DC in one family. The present report includes descriptions of diagnosis and treatment of family members diagnosed for the first time as having a DC. The report shows an importance of all diagnostic stages: from a medical history and clinical picture to an application of modern high-tech diagnostic methods (flow-FISH, NGS). The report underlines an importance of diagnosis of all family members for excluding an asymptomatic form after a case of DC has been already detected in that family. A high frequency of a toxicity and secondary neoplasia makes it necessary to realize an individual approach at treatment of each patient with DC (the earliest start of androgen treatment, prompt decision of implementation of allogenic hematopoietic stem cell transplantation). The knowledge of pathogenesis, clinical features and principles of diagnosis and therapy of this disease is relevant to pediatricians and hematologists.

Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.

Background Myelodysplastic syndromes (MDS) can present a challenge for clinicians. Multicolor flow cytometry (MFC) can aid in establishing a diagnosis. The aim of this study was to determine the optimal MFC approach for MDS. Methods The study included 102 MDS (39 low-grade MDS), 83 cytopenic patients without myeloid neoplastic disorders (control group), and 35 healthy donors. Bone marrow was analyzed using a six-color MFC. Analysis was conducted according to the "Ogata score," "Wells score," and the integrated flow cytometry (iFC) score. Results The respective sensitivity and specificity values were 77.5% and 90.4% for the Ogata score, 79.4% and 81.9% for the Wells score, and 87.3% and 87.6% for the iFC score. Specificity was not 100% due to deviations of MFC parameters in the control group. Patients with paroxysmal nocturnal hemoglobinuria (PNH) had higher levels of CD34(+)CD7(+)myeloid cells than donors. Aplastic anemia and PNH were characterized by a high proportion of CD56(+)cells among CD34(+)precursors and neutrophils. The proportion of MDS-related features increased with the progression of MDS. The highest number of CD34(+)blasts was found in MDS with excess blasts. MDS with isolated del(5q) was characterized by a high proportion of CD34(+)CD7(+)cells and low granularity of neutrophils. In 39 low-grade MDS, the sensitivities were 53.8%, 61.5%, and 71.8% for Ogata score, Wells score, and iFC, respectively. Conclusion The results support iFC as a useful diagnostic tool in MDS.

Background. Aplastic anemia (AA) is a disorder characterized by pancytopenia, hypoplastic bone marrow (BM), and the absence of underlying malignancy. It is believed to be of autoimmune nature. However, some patients fail to respond to the immunosuppressive therapy. The impaired hematopoietic microenvironment could be another reason for BM failure. The severity of AA varies widely from mild, chronic pancytopenia to total hematopoietic failure. The diagnosis of severe (SAA) and non-severe AA (NAA) is based on an absolute neutrophil count as an essential criterion. The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in BM of untreated SAA and NAA patients. Methods. The study included 22 AA patients (8 with SAA and 14 with NAA) in the debut of the disease. In all patients BM was aspirated after informed consent at diagnostic punctures. The proportion of non-hematopoietic CD45-CD34-CD71-CD235-CD90+CD73+CD105+ cells was estimated by FACS. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. Individual CFU-F-derived colonies were analyzed for their proliferative and differentiation potential. Adipogenic and osteogenic differentiation potential was analyzed with standard techniques. Relative expression level (REL) of several genes had been estimated with RT-PCR in Taqman modification. As a control 19 BM samples of healthy donors of according age were used. Results. The data are presented in the table. The proportion of non-hematopoietic cells was higher in the BM of AA patients than in healthy donors. We recalculated the proportion of CFU-F among non- hematopoietic cells; it was similar in the BM of AA patients and healthy donors. However, the concentration of -CFU-F was much higher in the BM of patients with SAA then in the BM of patients with NAA. Among NAA patients, 2 had PNG clone and unlike other NAA patients increased CFU-F concentration, comparable to patients with SAA. It seems that the character of stromal cell damage depends on the severity of AA. Individual CFU-F- derived clones from the BM of NAA patients had very limited proliferative potential, while those of SAA patients did not differ from colonies of healthy donors. The analysis of CFU-F-derived colonies differentiation ability revealed that the proportion of the precursors that did not respond to the differentiation induction was higher in the BM of AA patients than in donors. It reflects the involvement of a certain subpopulation of stromal precursors that are either pre-differentiated into fibroblasts, or, conversely, earlier precursors of the hematopoietic microenvironment, which were not able to differentiate into osteoblasts and/or adipocytes within standard time. The analysis of the MMSC growth characteristics revealed that the time required for MMSC from SAA and NAA patients to form a confluent monolayer after the initial seeding and the population doubling time, were significantly higher than in MMSC of healthy donors. Thus, the proliferation rate of MMSC of AA patients is reduced. Nevertheless, the total cell production for 3 passages did not differ in cultures of AA patients and healthy donors. Therefore, the proliferative potential of MMSC of AA patients is not altered. Probably MMSC being analyzed ex vivo can restore their function. However, the analysis of REL of genes regulating the proliferation (FGF2, FGFR1, FGFR2) in MSCs had revealed the differences in comparison with donors and between SAA and NAA. Moreover, the analysis of the polymorphism in CFH gene, participating in immunomodulation, showed that the distribution differs between NAA and SAA patients. Conclusions. Stromal precursors in BM of untreated NAA and SAA patients are impaired and differ between the two subtypes of AA. It seems that the differences between NAA and SAA may lay not only in the absolute neutrophil count but also in the BM stroma itself. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the hematopoiesis failure. This work was supported by the Russian Foundation for Basic Research, project no. 19-015-00280. Table Disclosures No relevant conflicts of interest to declare.

Introduction. The main pathogenetic mechanism of the development of aplastic anemia (AA) is a violation of the immune regulation of hematopoiesis.Aim: to study of the subpopulation composition of T-cells and the repertoire of the T-cell receptor in AA patients.Patients and Methods. The study included AA patients (n = 40) without prior immunosuppressive therapy in 2018–2020. The T-cell subpopulation structure and T-cell receptor Vβ-family (TCR-Vβ) oligoclonality were studied in samples of bone marrow using flow cytometry.Results. We report characteristic properties of T-cell subpopulations of bone marrow in all AA patients: elevated counts of cytotoxic T-cells, effector CD4+ and CD8+ cells, CD4+ memory cells, which may suggest a long-term antigenic stimulation with subsequent activation of these cell subpopulations resulting in hyperexpression of pro-inflammatory cytokines. Diminishing of naive CD4+ and CD8+ cells, regulatory and double negative T-cells may indicate a relaxing control of cytokine-producing T-cells. A relationship has been established between the AA severity and counts of effector, regulatory, double negative and PD-1 positive T-cells. A highest count of potentially cytokine-producing T-cells and lowest count of cells involved in T-cell activity regulation were observed in very severe AA patients. Studies of the TCR-Vβ repertoire revealed oligoclonal expansion in the cytotoxic T-cell subpopulation.Conclusion. Enrichment in selected Vβ families suggests autoreactive T-cell clonality and attests to the immune nature of AA. A dynamic TCR-Vβ repertoire assay may be recommended in the disease monitoring. Flow cytometry helps identify valuable biomarkers for T-cell clone monitoring in AA and a better assessment of the disease progression.

Background . Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal diseases of the hematopoiesis system characterized by dismyelopoiesis and cytopenia, the presence of cytogenetic aberrations and a high risk of transformation into acute myeloid leukemias. Diagnosis of MDS requires a comprehensive approach and mandatory performance of cytological, cytochemical and cytogenetic studies of bone marrow aspirate, as well as histological examination of trephine biopsy. However, in some cases it is necessary to undergo a diagnostic test that would allow verification of the MDS. The study of bone marrow aspirate by multicolor flow cytometry (MFC) can be considered as an additional diagnostic criterion in the diagnosis of MDS.The objective of the study was to estimate the incidence of myelodysplastic features in patients with various forms of MDS by the MFC method. Materials and methods . The study included 79 patients with MDS: 8 with MDS with 5q deletion, 33 with MDS without excess blast cells and 38 with excess of blasts. A bone marrow aspirate test was performed by 6-color flow cytometry. The control group included 35 donors of allogeneic bone marrow. The analysis resulted in a conclusion on the Ogata score scale, the Wells prognostic scale and the combined Ogata–Wells scale. When using the screening method, the presence of two or more cytometric signs of MDS was detected in 60 (75.9 %) of 79 MDS patients. Wells score was higher in MDS group with an excess of blast than in others. Using the combined Ogata–Wells scale, cytometric aberrations were found in 70 (88.6 %) of 79 MDS patients. In patients with MDS with an excess of blasts, the incidence of increased CD34+ and/or CD117+ myeloid cells was higher than in MDS patients without an excess of blasts and an MDS with a 5q deletion. The frequency of abnormal cytometric parameters (anomalous expression of CD34, CD117, CD56+ myeloblasts) in these groups did not differ. In patients with isolated 5q deletion and MDS without excess of blasts, an increased proportion of CD7+CD34+ cells was more often detected than in MDS with an excess of blasts.Conclusion . Thus, cytometric abnormalities in MDS are common, even in patients without excess of blasts. The MFC method can be used as an additional diagnostic method in the initial diagnosis of MDS.

Myelodysplastic syndromes (MDS) require a comprehensive approach in the primary diagnostics. Multicolor flow cytometry (MFC) can be as an additional diagnostic tool but is not very standardized.

Immunosuppressive therapy (IST) proved to be effective in 70-90% of AA patients. However, not all the questions are solved, particularly, treatment of patients with AA refractory to IST without HLA-compatible donor. The purpose of the study is optimization of treatment algorithm in this group of patients with AA.

The aim of the study was to evaluate the frequency of Human Herpes Virus 6 (HHV6) infection in immunocompromised patients and to analyze the efficiency of the antiviral treatment. In retrospective study 273 patients with hematological disorders were analyzed from Jan 2013 to Jul 2017 (72 patients with acute lymphoblastic leukemia (ALL), 141 case - with acute myeloid leukemia, 15 cases with high-grade B-cell lymphoma (BCL), 45 aplastic anemia patients. 25 patients underwent autologous stem cell transplantation (19 with ALL, 6 with BCL). DNA of HHV6 was detected in 32.9% of patients, including 11 patients after transplantation. However, the detection of HHV6 with clinical symptoms was identified only in 26 (9.5%) of 273 patients. Febrile neutropenia, hemorrhagic cystitis, enteropathy, pneumonia, 1 case of encephalitis and 1 case of severe cutaneous HHV6 lesion were detected as clinical manifestations of the infection. The importance of prolonged intravenous antiviral treatment was demonstrated. Thus, HHV6 DNA is detected in large percent of cases in hematological patients but its clinical manifestation is not seen very often. The early prolonged and adequate treatment reduces the risks of complications.