[Thrombosis in patients with myeloproliferative neoplasms. Case report].

Thrombotic complications are the most significant factors determining the prognosis in myeloproliferative neoplasms. Markers for assessing the risk of thrombosis are the number of leukocytes, platelets, hemoglobin level, hematocrit, age, molecular status, history of thrombosis, obesity, arterial hypertension, hyperlipidemia, hereditary or acquired thrombophilia. The pathogenesis of thrombosis in patients with myeloproliferative neoplasms is complex and multifactorial. In most cases, the etiological factor remains unknown. Currently, antiplatelet and anticoagulant therapy is carried out on an individual basis. The algorithm for primary and secondary (after thrombosis) prevention requires development and testing. We present a clinical case of repeated arterial and venous thrombotic complications in a patient with primary myelofibrosis.

[The prognostic value of ASXL1 mutation in primary myelofibrosis. Literature review and clinical case description].

Primary myelofibrosis is a myeloproliferative neoplasm that occurs de novo, characterized by clonal proliferation of stem cells, abnormal expression of cytokines, bone marrow fibrosis, hepatosplenomegaly as a result of extramedullary hematopoiesis, symptoms of tumor intoxication, cachexemia, peripheral blood leukoerythroblastosis, leukemic progression and low survival. Primary myelofibrosis is a chronic incurable disease. The aims of therapy: preventing progression, increasing overall survival, improving quality of life. The choice of therapeutic tactics is limited. Allogenic hematopoietic stem cell transplantation is the only method that gives a chance for a cure. The role of mutations in a number of genes in the early identification of candidates for allogeneic hematopoietic stem cell transplantation is being actively studied. The article describes the clinical case of the detection ofASXL1gene mutations in a patient with prefibrous primary myelofibrosis. The diagnosis was established on the basis of WHO criteria 2016. The examination revealed a mutation ofASXL1. Interferon alfa therapy is carried out, against the background of which clinico-hematological remission has been achieved. Despite the identified mutation, the patient is not a candidate for allogeneic hematopoietic stem cell transplantation. Given the unfavorable prognostic value of theASXL1mutation, the patient is subject to active dynamic observation and aggressive therapeutic tactics when signs of disease progression appear.

[Clinical features and diagnosis of Ph - negative myeloproliferative neoplasms occurring in conjunction with the antiphospholipid syndrome].

Thrombosis is a serious and extremely dangerous disease that has a negative impact on the quality and longevity. Antiphospholipid syndrome (APS) is a pathology characterized by recurring venous, arterial, microvasculature thrombosis, pregnancy pathology with loss of the fetus and the synthesis of antiphospholipid antibodies. A high risk of thrombotic complications is also observed in patients with myeloproliferative neoplasms (MPN). This article presents a description of three clinical cases of Ph - negative myeloproliferative diseases, occurring in conjunction with APS. In all cases, recurrent thrombosis allowed to suspect the presence of two diseases - MPN and APS.

[Thrombotic events in patients with hemophilia]. / Tromboticheskie oslozhneniia u bol'nykh gemofiliei.

The paper describes 4 clinical cases of thrombotic events (pulmonary embolism, deep vein thrombophlebitis, acute myocardial infarction, ischemic stroke) that have occurred in patients with hemophilia. It discusses the possible causes of their development and methods for their prevention and treatment. Controlled natural hypocoagulation, in which the dose of an administered deficient factor decreases to such an extent that in order to maintain the safe level of hypocoagulation (plasma factor activity is 15-20%; activated partial thromboplastin time is 1.5-2 times normal values), is proposed as one of the treatment options.

[Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots].

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

[First experience of using modified high-dose therapy NHL-BFM-90 in diffuse large B-cell lymphosarcoma with primary skin involvement. A case report].

Primary skin large B-cell lymphosarcomas (PLBCL) present with skin lesions, other organs and systems are not involved. As CHOP courses are not high effective in PLBCL, we were the first to treat a patient with modified block therapy NHL BFM-90. A complete remission was achieved after the first course of polychemotherapy and was consolidated by two courses of treatment. Further follow-up is needed.

[A morphometric analysis of the nuclei and nucleoli in tumor cells in lymphogranulomatosis, diffuse large B-cell lymphoma and anaplastic large cell lymphoma].

AIM: To make a comparative morphometric analysis of the nuclei and nucleoli of tumor cells in lymphogranulomatosis (LGM), diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL) for differential diagnosis of these lymphomas. MATERIAL AND METHODS: Biopsy material (lymph node biopsies) was frozen in hexane, fixed and stained, then microscopic pictures were made. RESULTS: Mean area of tumor cell nuclei in LGM was 97.25 +/- 68.77 mcm2, in DLBCL and ALCL--55.89 +/- 20.13 mcm2 and 70.31 +/- 34.64 mcm2, respectively. The area differences were significant (p < 0.001). Hodgkin's and Berezovsky-Rid-Sternberg cell bucleoli area was the largest (11.44 +/- 7.83 mcm2). The nucleoli of the former are larger than those of the latter. Mean area of the nucleoli in DLBCL was 3.05 +/- 1.58, in ALCL--5.53 +/- 4.94 mcm2. The differences are significant (p < 0.001). CONCLUSION: Nucleoli in Hodgkin 's cells are significantly larger than those in the tumor cells in ALCL and DLBCL and the nucleoli with the area more than 12 mcm2 can be used in differential diagnosis between LGM and DLBCL but not between LGM and ALCL.

[Efficacy of therapy of different variants of anaplastic large T-cell lymphomas].

AIM: To compare efficacy of NHL-BFM-90 and CHOP-like courses in the treatment of anaplastic large cell lymphoma (ALCL). MATERIAL AND METHODS: Twenty-two patients with ALCL participated in the study. The diagnosis was made basing on the findings of clinical, device, morphological, immunohistochemical and molecular-genetic examinations with application of a panel of monoclonal antibodies to CD30, ALK, CD3, CD4, CDS, CD7, CD34, CD15, CD68, CD20, CD45RO, CD45RA, Ki-67. 14 cases of 22 were negative by kinase of anaplastic lymphocytes (ALK-) and 8 were positive (ALK+). Mean age of ALK-ALCL patients was 39.6 +/- 4.1 years, of ALK+ALCL patients - 23.4 +/- 2.6 years. 14 patients were treated by the protocol NHL-BFM-90, 8 were initially treated with other schemes (CHOP, MACOP-B, BEACOPP and others). All 14 patients treated according to NHL-BFM-90 had ALCL stages III-IV with B-symptoms. 12 patients who completed treatment by the above protocol achieved complete remission after the forth course, 2 patients failed the treatment. Of 8 ALCL patients treated initially according to other schemes, a complete remission was achieved in 4 patients (2 had stage II). One of 4 patients with remission had recurrence. Four patients who had failed to achieve complete remission died of the disease progression. CONCLUSION: ALCL occurs more frequently in young and middle-aged patients. The disease has an aggressive course with rapid generalization. For such processes it is more preferable to use a modified protocol NHL-BFM-90.

[Flow fluorimetry in differential diagnosis of diffuse large B-cell lymphoma].

AIM: To analyse immunophenotype of diffuse large B-cell lymphoma (DLBCL) with flow cytometry. MATERIAL AND METHODS: Combinations of antibodies against the following antigens were used: CD3/ CD19/CD45, CD5/CD19/CD38, CD19/CD10/CD23, CD4/CD8/CD3, kappa/lambda/CD19, CD25/CD20/FMC7; CD43/CD22/CD20; CD79a/Ki-67/CD3; cytoplasmic kappa/lambda. The analysis was made on flow fluorimeter FacsCalibur using computer program CellQuest (Beckton Dickenson, USA). RESULTS: Specific coexpression of markers is not detectable in DLBCL, in the greatest degree the phenotype corresponds to lymphoma from the cells of the marginal zone. The study of cells with maximal direct light diffusion provides more precise assessment of clonality and proliferative potential of tumor cells than the analysis of the whole lymphocytic polygon. The proliferative index in 33 cases of DLBCL varied in the range 10-60%. CONCLUSION: Flow cytometry in most DLBCL cases allows identification of B-cell clonality, more precise assessment of a proliferative potential of the tumor.

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.

The role of Ca ions in restoration of the structure of interphase and mitotic chromosomes in PK living cells after hypotonic stress.

The dynamics of mitotic chromosome and interphase chromatin recondensation in living PK cells during their adaptation to hypotonic medium was studied. The recondensation process was found to be slowed down by the modification of plasma membrane with low concentrations of glutaraldehyde, while osmotic reactions of glutaraldehyde-treated cells remain unchanged. The effect of glutaraldehyde can be rapidly reversed by the addition of Ca(2+)-ionophore A23187. Intracellular Ca(2+)measurements show that the adaptation to hypotonic shock is accompanied by restoration of free Ca concentration, whereas the delay of chromatin condensation in glutaraldehyde-treated cells is paralleled by the decrease of Ca level. The mechanisms implying the role of low concentration of Ca(2+)in chromatin compactization in vivo are discussed.

Blue light inhibits mitosis in tissue culture cells.

Irradiation of the mitotic (prophase and prometaphase) tissue culture PK (pig kidney embryo) cells using mercury arc lamp and band-pass filters postponed or inhibited anaphase onset. The biological responses observed after irradiation were: (i) normal cell division, (ii) delay in metaphase and then normal anaphase and incomplete cytokinesis, (iii) exit into interphase without separation of chromosomes, (i.v.) complete mitotic blockage. Cell sensitivity to the light at wavelengths from 423 and 488 nm was nearly the same; to the near UV light (wavelength 360 nm) it was 5-10 times more; to the green light (wavelength >500 nm) it was at least 10 times less. To elucidate the possible mechanism of the action of blue light we measured cell adsorption and examined cell autofluorescence. Autofluorescence of cytoplasmic granules was exited at wavelengths of 450-490 nm, but not at >500 nm. In mitotic cells fluorescent granules accumulated around the spindle. We suppose blue light irradiation induces formation of the free radicals and/or peroxide, and thus perturb the checkpoint system responsible for anaphase onset.

Centrosome and microtubules behavior in the cytoplasts.

After enucleation of PK (pig kidney embryo) cells using cytochalasin D centrioles remained approximately in 80% of cytoplasts. Some cytoplasts retained a single centriole. 14-16 h after enucleation large secondary lysosomes and lipid droplets were evident around the centrosome of many cytoplasts. In part of the cytoplasts replicating centrioles were found 16 h after enucleation. Ouabain treatment (1 mM, 30 min) of the cytoplasts resulted in the appearance of mainly perpendicular orientation of mother centrioles relative to the substrate surface. Reconstruction of microtubule pattern around the centrosome showed that a total of approximately 15 microtubules were attached in the centrosome of normal cells and twice more than that in cytoplasts. Microtubules in the cytoplasts were more resistant to nocodazole induced depolymerization. We suggest that microtubule instability is modulated by regulatory effector that is under direct nucleus control.

[Centrioles can replicate in cultured cells in the absence of a nucleus]. / Tsentrioli mogut replitsirovat'sia v kul'tiviruemykh kletkakh v otsutstvie iadra.

Cytoplasts of cultured L fibroblasts, obtained by enucleation at the end of G1 of synchronized cells collected in mitosis, have usually two centrioles. Starting from 8 h after enucleation, the centrioles in all cytoplasts were seen replicating. The formed procentrioles had an average length of 0.2 micron and did not grow within the next 16 h. When the same synchronized L cells were treated from the end of G1 with actinomycin D, inhibiting more than 96% of RNA synthesis, procentrioles were not observed for about 16 h of incubation. It is concluded that centrioles in cultured cells could replicate in the absence of RNA synthesis, and that the nucleus may not only regulate the centriole replication, but also suppress this process.

[The ultrastructure of the centrosome in cytoplasts and its alteration under the action of ouabain]. / Ul'trastruktura tsentrosomy v tsitoplastakh i ee izmenenie pod deistviem uabaina.

It has been shown that after enucleation of the PE cells with cytochalasin D the centrioles remain in approximately 80% of cytoplasts. Some cytoplasts contain only single centriole, either a mother (active) of a daughter (inactive) one. 20% cytoplasts have no centrioles. 2h after enucleation the centrosome structure in the cytoplasts did not differ from that in normal cells. 14-16 h after enucleation in many cytoplasts large secondary lysosomes and lipid droplets appeared around the centrosome. At this time in some cytoplasts in the centrosome we observed free microtubule convergence foci. 14-16 h after the enucleation, some cytoplasts have doubling centrioles. Under the influence of ouabain (30 min), the number of active centrioles oriented perpendicularly to the substrate plane in the cytoplasts increased. We suggest that the preferentially perpendicular orientation of centrioles to the substrate plane does not depend on the nuclear activity.

[The inhibition of mitosis in tissue culture cells by blue light]. / Ingibirovanie mitoza v kletkakh kul'tury tkani sinim svetom.

Blue light (wavelength 350-480 nm) irradiation of the early mitotic (prophase and prometaphase) tissue culture cells at the dose of 50-3000 J/cm2 delay mitosis or completely block it at the metaphase. Cell sensitivity to the near UV light (wavelength 360 nm) was few times more as compared with the sensitivity to the visible light (wavelength 400-480 nm). Mitotic cells irradiated with the green light (wavelength more than 500 nm; dose up to 7500 J/cm2) completed division normally. The effect of the blue light did not depend on the presence of phenol red in tissue culture medium. Rhodamin 123 staining did not show any changes in the mitochondrial system in the irradiated mitotic cells. Blue light irradiation with the dose enough for the induction of mitotic delay appears to be insufficient to affect the proliferation of interphase cells.