Number Of Megakaryocytes Research Articles

Mice Lacking α-Actinin-1 in Megakaryocytes Display the Feature of Thrombocytopenia and Impair Platelet Functions

Cytoskeleton remodeling and mitochondrial bioenergetics play important roles in platelet biogenesis and functions. α-Actinin-1, a filament crosslinker, was widely expressed in nonmuscle cells, including megakaryocytes (MKs) and platelets. The cytoskeleton is essential for normal mitochondrial morphology, motility, and distribution. In recent years, the accumulated evidence suggests that inherited mutations in α-actinin-1 are implicated in congenital macrothrombocytopenia. However, the role and underlying mechanism of α-actinin-1 in platelet biogenesis and platelet functions remain poorly studied. In this study, we generated an MK-specific α-actinin-1 knockout mouse model (referred to herein as PF4- Actn1-/-) to investigate the contribution of α-actinin-1 in platelet biogenesis and functions. Blood count tests showed that PF4- Actn1-/- mice exhibited reduced platelet counts. The decreased platelet number in PF4- Actn1-/- mice was not attributed to accelerated platelet clearance or impaired TPO generation in vivo. Platelet count changes observed in an anti-GPIbα antibody-induced thrombocytopenia model and splenectomy indicated that the diminished platelet counts in PF4- Actn1-/- mice might be due to defects in platelet biogenesis. Compared to control mice, PF4- Actn1-/- mice had normal numbers of MK progenitors, including in LSK, MPP, CMP, GMP, MEP, and PreMegE. H&E staining and flow cytometry revealed a decrease in the number of MKs in BM, but an increase in the number of MKs in the spleen. The absence of α-actinin-1 significantly increased the proportion of 2N-4N MKs and decreased the proportion of 8N-32N MKs. CFU-MK colony formation and MK migration in response to SDF-1 signaling was inhibited in PF4- Actn1-/- mice. Furthermore, in vitro studies showed a reduced ratio of PPF-bearing MKs in fetal liver-derived PF4- Actn1-/- MKs. Collectively, these data suggest that α-actinin-1 deficiency in mouse MKs could impair platelet biogenesis, resulting in thrombocytopenia in PF4- Actn1-/- mice. Platelet adhesion and spreading on immobilized fibrinogen and collagen, thrombin-stimulated clot retraction, and platelet aggregation in response to various concentrations of ADP, thrombin, and collagen was significantly decreased in PF4- Actn1-/- platelets. PF4- Actn1-/- platelets also exhibited decreased integrin αIIbβ3 activation and reduced P-selectin exposure in response to various concentrations of ADP, ADP+EPI, thrombin, collagen, and convulxin. Notably, PF4- Actn1-/- platelets showed inhibited calcium mobilization, reactive oxygen species (ROS) generation, and actin polymerization in response to collagen and thrombin. Furthermore, PF4- Actn1-/- mice demonstrated significantly impaired hemostasis. No differences were observed in the PT, APTT, TT, and fibrinogen concentrations between PF4- Actn1-/- mice and Actn1f/f mice. However, tail, liver, and brain bleeding tests revealed that PF4- Actn1-/- mice had significantly prolonged bleeding time and increased bleeding volume. Kaolin-activated whole thromboelastography (TEG) mapping assay indicated that there were no differences in the R-time, K-time, and α-angle values, but the maximum amplitude was decreased in PF4- Actn1-/- mice. To explore the mechanism of α-actinin-1 in platelet biogenesis and platelet functions. Low- (2-4N) and high-ploidy (≥8N) Actn1f/f and PF4- Actn1-/- MKs were sorted for quantitative proteomics analysis. The results of 4D label-free quantitative proteomics revealed that protein expression was significantly decreased in high-ploidy MKs following α-actinin-1 knockdown. Functional enrichment analysis of differentially expressed proteins indicated that α-actinin-1 deletion reduced platelet activation and mitochondrial functions. PF4- Actn1-/- platelets exhibited reduced mitochondrial membrane potential, mitochondrial ROS generation, mitochondrial respiration and glycolysis, suggesting alterations in mitochondrial function in platelets. In this study, we report that mice with α-actinin-1 deficiency in MKs recapitulate the features of thrombocytopenia and exhibit impaired platelet functions.

Integrin αvβ8-Mediated Activation of TGF-β1 Is Involved in Regulating the Sustained Response after TPO-Ras Withdrawal in ITP Patients

Background: Thrombopoietin receptor agonists（TPO-RAs）are effective and safe in immune thrombocytopenia (ITP), and are currently the most important second-line drugs. The effect of TPO-RAs is transient, platelet counts usually return to baseline within 2 weeks unless treatment is continued. Only one-third of patients can maintain after drug discontinuation, but the mechanism is unknown and worthy of further exploration. Method and Results：In this study we observed 66 patients treated with TPO-RAs and followed-up to three months after drug withdrawal. Patients were divided into three groups: non-response group (NR), sustained group (Group A), and non- sustained group (Group B) (Fig1-A). The blood samples were collected at the drug discontinuation, and we found that compared with total TGF-β1 levels，plasma activated TGF-β1 levels were extremely lower in NR patients and were also reduced in group B (Fig1-B,C). At the same time, regulatory T cells have the same trend. By using the qPCR and Western blot we preliminary found that integrin αvβ8 regulate the activation of TGF-β1 to promote the formation of Tregs, which affects the sustained response to TPO-RAs withdrawal. Furthermore, lower levels of activated TGF-β1 in the bone marrow were associated with higher numbers of megakaryocytes, fewer plate-producing megakaryocytes, and decreased proportion of hyperploid megakaryocytes (≥16N). Through in vitro cell culture, we found that recombinant human integrin avb8 protein can promote Treg differentiation and inhibit T cell activation in ITP patients' PBMCs, but this phenomenon can be reversed by anti-β8 antibody. Then, we constructed PF4-TGF-β1 conditional knockout mice (Cre+ mice) and constructed of an ITP passive model by injecting CD41 antibody and found that the Cre+ mice have higher platelets at baseline, while the platelets dropped rapidly than Cre- mice after CD41 antibody injection (Fig2-A). Using rhTPO to treat the ITP mice, we found that Cre+ mice were less effective than Cre- mice, and the sustained response time was shorter after drug withdrawal. When sacrificed the mice, decreased Treg cells and elevated M1 macrophages were detected in Cre+ mice. Meanwhile, HE staining of bone marrow found a greater number of megakaryocytes, but the cell diameter decreased significantly (Fig2-B, D). Next, we treated the mice with rhTPO in combined with recombinant mice integrin avb8 (50ng/kg or 250ng/kg), the therapeutic efficacy and sustained response time in the high-dose avb8 group were significantly improved (Fig2-C) We also constructed a more representative ITP active model of autoimmune diseases, by immunizing CD61KO mice to produce autoantibodies and infusing spleen cells back into SCID mice. Simultaneously through transfection siRNA or shRNA to activate or silence mouse T cell integrin β8 and reinfuse it into SCID mice. We found that activating integrin β8 in mice had a more significant therapeutic effect and maintained a longer response time. Finally, through single-cell sequencing analysis, the smads signaling pathways，integrin binding pathways and AP-1 family signaling pathways were significantly enriched in group A patients. Further study confirmed that TPO-RAs can regulate the AP-1 family, which upregulated the β8 subunit, resulting in enhanced activation of TGF-β1 (Fig3-A, B). Conclusion: Integrin αvβ8 activates TGF-β1 and upregulates Tregs to restore immune intolerance in ITP patients, meanwhile, activated TGF-β1 also promotes hyperploidy megakaryocyte differentiation in bone marrow, which co-regulates the sustained response after TPO-RAs discontinuation.

Evaluation of the Histopathological Changes Accompanied for the Toxic Effects of Diazinon on the Spleen of Mice

This study was carried out to determine the histopathological changes caused by diazinon in the spleen of Swiss albino mice. Experimental groups containing low dose (30 mg/kg), medium dose (60 mg/kg) and high dose (120 mg/kg) were exposed to diazinon through oral administration for 30 consecutive days. Separation and hemorrhage in the capsule, congestion, enlarged white pulp, amyloid formation, and karyolysis in some megakaryocytes were determined in the splenic parenchyma of the low dose group. An increase in the number of enlarged white pulps, hemorrhage within splenic parenchyma, accumulation of cells into dilated sinusoids and amyloid formation were examined in the medium dose group. Some cells passing from the splenic parenchyma into dilated sinusoids were also observed. Intensive congestion, necrotic areas within spleen tissue, an increase in the number of karyolytic megakaryocytes, fibrosis and some cells passing from the splenic parenchyma into enlarged sinusoids were prominent histological lesions in the high dose group. These results showed that diazinon caused severe dose-related histopathological damages and had the capacity to disrupt functions of the spleen.

Multiphoton In Vivo Microscopy of Embryonic Thrombopoiesis Reveals the Generation of Platelets through Budding.

Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production.

The role of PKC in X-ray-induced megakaryocyte apoptosis and thrombocytopenia

Thrombocytopenia is a critical complication after radiation therapy and exposure. Dysfunction of megakaryocyte development and platelet production are key pathophysiological stages in ionizing radiation (IR)-induced thrombocytopenia. Protein kinase C (PKC) plays an important role in regulating megakaryocyte development and platelet production. However, it remains unclear how PKC regulates IR-induced megakaryocyte apoptosis. In this study, we found that pretreatment of PKC pan-inhibitor Go6983 delayed IR-induced megakaryocyte apoptosis, and inhibited IR-induced mitochondrial membrane potential and ROS production in CMK cells. Moreover, suppressing PKC activation inhibited cleaved caspase3 expression and reduced p38 phosphorylation levels, and IR-induced PKC activation might be regulated by p53. In vivo experiments confirmed that Go6983 promoted platelet count recovery after 21 days of 3 Gy total body irradiation. Furthermore, Go6983 reduced megakaryocyte apoptosis, increased the number of megakaryocyte and polyploid formation in bone marrow, and improved the survival rate of 6 Gy total body irradiation. In conclusion, our results provided a potential therapeutic target for IR-induced thrombocytopenia.

Natural history study of patients with familial platelet disorder with associated myeloid malignancy

AbstractDeleterious germ line RUNX1 variants cause the autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematologic malignancies (HMs). We launched a FPDMM natural history study and, from January 2019 to December 2021, enrolled 214 participants, including 111 patients with 39 different RUNX1 variants from 45 unrelated families. Seventy of 77 patients had thrombocytopenia, 18 of 18 had abnormal platelet aggregometry, 16 of 35 had decreased platelet dense granules, and 28 of 55 had abnormal bleeding scores. Nonmalignant bone marrows showed increased numbers of megakaryocytes in 12 of 55 patients, dysmegakaryopoiesis in 42 of 55, and reduced cellularity for age in 30 of 55 adult and 17 of 21 pediatric cases. Of 111 patients, 19 were diagnosed with HMs, including myelodysplastic syndrome, acute myeloid leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and smoldering myeloma. Of those 19, 18 were relapsed or refractory to upfront therapy and referred for stem cell transplantation. In addition, 28 of 45 families had at least 1 member with HM. Moreover, 42 of 45 patients had allergic symptoms, and 24 of 30 had gastrointestinal (GI) symptoms. Our results highlight the importance of a multidisciplinary approach, early malignancy detection, and wider awareness of inherited disorders. This actively accruing, longitudinal study will genotype and phenotype more patients with FPDMM, which may lead to a better understanding of the disease pathogenesis and clinical course, which may then inform preventive and therapeutic interventions. This trial was registered at www.clinicaltrials.gov as #NCT03854318.

Bone marrow composition features and possibilities of tumor involvement diagnosing in patients with classic Hodgkin’s lymphoma

Background. Classical Hodgkin’s lymphoma is a B-cell lymphoproliferative disease, the tumor substrate of which is Berezovsky–Reed–Sternberg cells, characterized by CD30, PAX-5, CD15 expression and the absence of CD3, CD45. In some cases, tumor cells express CD20. modern anticancer therapy has increased the survival probability for most patients, not only with early but also with advanced stages of classical Hodgkin’s lymphoma. Such successes are mainly due to the distribution of patients into prognostic groups and the choice of an appropriate treatment regimen. tumor infiltration of the bone marrow suggests assigning patients to the advanced stages group, followed by the choice of an intensive therapy program. Aim. To determine the bone marrow involvement frequency according to positron emission tomography combined with computed tomography (PET/CT), with 18F-fluorodeoxyglucose (FDG) and bone marrow trephine biopsy (bmtb), to compare the results obtained with primary tumor immunophenotype and bone marrow cellular composition, and to identify of prognostic risk factors. Materials and methods. The study included 107 patients with newly diagnosed classical Hodgkin’s lymphoma, who underwent a diagnostic examination at the moscow research institute of oncology named after P. A. Herzen – a branch of the National Medical Research Center for Radiology and the “Lapino” clinical Hospital from 2015 to 2022, followed by anticancer therapy and further follow-up. Morphology of the primary tumor biopsy specimen in all patients and immunohistochemical (IHC) study using a wide panel of monoclonal antibodies (CD15, CD30, CD3, CD45, CD20, PAX-5 anti-gens; in some cases epstein–barr virus proteins expression) in most cases were performed. All patients underwent a morphological and / or IHC study of BMTB and the majority underwent aspiration biopsy and PET/CT with 18F-FDG. Results. The most common histological variant of classical Hodgkin’s lymphoma was nodular sclerosis (86.9 %). The majority of patients (51.4 %) were assigned to the advanced stage prognostic group. bone marrow tumor infiltration was statistically significantly more frequently diagnosed during PET/CT with 18F-FDG compared with the results of the BMTB – in 27.1 % and 12.1 % of cases, respectively (p < 0.05). when comparing the results of both diagnostic methods, it was found that in 17.1 % of cases, bone marrow infiltration, detected during PET/CT, was not confirmed by IHC examination of the trephine biopsy. In addition, it was found that the majority of cases with CD20+/± and CD15+/± expression in the primary tumor were observed in the group of patients without bone marrow involvement. when assessing the cellular composition of bone marrow aspirates, it was revealed that in patients with bone marrow tumor infiltration, an increase in cellularity and megakaryocytes number along with a decrease in the plasma cells number is observed. Conclusion. The results suggest further study of bone marrow immunomorphological features in order to identify prognostic factors and search for new therapeutic targets. a more extended analysis of bone marrow aspirate immunomor-phological characteristics using new modern diagnostic methods, minimal residual disease status as a surrogate marker seems to be relevant and necessary to confirm the depth of the antitumor response achieved. Detection of CD20+/±- and cd15+/±-Berezovsky–Reed–Sternberg cells according to primary tumor IHC analysis indicates a low probability of bone marrow tumor infiltration, but further analysis is required on a large clinical and laboratory material.

Purification of Megakaryocytes for flow cytometric analysis

Objectives: Megakaryocytes (MKs) represent only a limited cell number in normal bone marrow, which hampers the analysis of MKs by flow cytometric technique. To optimize the usage of flow cytometry for the analysis of megakaryopoiesis, we established a simple method to enrich MKs. Methods: Bone marrow tissues were taken from mice (n=5) and made into cell suspensions. The cell suspension was processed by velocity sedimentation with a continuous BSA solution (3%-6%) for 1h at room temperature from top to bottom of the gradient to purify MKs. Each layer (1 ml) gradient from top to bottom was collected and the number of MKs and other cells was counted under a microscope. The enriched MKs gradients were located in the lower 5 ml between layer 7 and layer 11 following sedimentation. They were then collected, centrifuged and labeled by antibody anti-CD41-FITC, and their DNA was also labeled by propidium iodide. Then, cells were performed isotype antibody labeling instead of CD41 for flow cytometric analysis. Another group underwent the same procedures at the beginning of cell suspension. Results: Under the microscope, The MKs were enriched mainly in the lower 7~11 layers, while other small cells were largely enriched in the upper 1~6 layer following sedimentation. The ploidy analysis by flow cytometric analysis showed that there was a typical peak which can represent various DNA content in the enriched MKs group, while the peak was not typical in the group without MK isolation. Conclusions: The results showed that the purification of MKs was effective in this experimental condition, providing a possibility to analyze megakaryopoiesis by flow cytometry instrument.

Myelofibrosis at diagnosis is associated with the failure of treatment-free remission in CML patients.

The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence.

Ameliorative Effects of Vitamins C and E on Haematotoxicity and Spleen Histopathology Induced by Dichlorvos Insecticide in Female Wistar Rats

This research aimed to assess the toxicological effects of dichlorvos insecticide (2,2-dichlorovinyl dimethyl phosphate or DDVP) on haematological indices of female Wistar rats and to investigate the antioxidant properties of vitamins C and E in ameliorating haematotoxicity. The rats were divided into 6 groups; control-water, control-oil, dichlorvos group, dichlorvos + vitamin C group, dichlorvos + vitamin E group and dichlorvos + vitamin C + E group. Dichlorvos and vitamins were orally administered to rats with an interval of half an hour between treatments. The control-oil group was given 2 ml corn oil and the control-water group was given water adlib. The treatments were done for 28 consecutive days and blood samples were taken by cardiac puncture. The following haematological parameters were analysed: RBC, WBC, Hb, PCV, MCV, MCH, MCHC and THR. Bone marrow smears were prepared on 14th and 28th days for various blood stem cells evaluation and spleen tissues for histopathology. There was a significant decrease (p ˂ 0.01) in RBC counts, PCV, MCV and MCH values, whereas the WBC, THR counts and MCHC values increased significantly (p ˂ 0.01) in dichlorvos treated rats as compared to the controls and dichlorvos + vitamins co-treated groups. There were no significant differences in blood parameter counts between controls and dichlorvos + vitamins co-treated groups. In the bone marrow smear of the dichlorvos treated group, there was increased number of megakaryocytes and mature neutrophils. In conclusion, findings from the current study revealed that, vitamins C and E supplements were capable of mitigating the haematotoxic effects induced by dichlorvos insecticide in Wistar rats.

High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3Δ/Δ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.

Severe systemic inflammation mimicking TAFRO syndrome following COVID-19.

TAFRO syndrome is a rare systemic inflammatory disease. Its pathogenesis mainly involves excessive cytokine secretion and autoimmune dysfunction. Although its etiology is unclear, some viral infections have been reported to cause it. Here, we report a case of severe systemic inflammation mimicking TAFRO syndrome that arose after COVID-19. A 61-years-old woman suffered from a continuous fever, ascites, and edema after contracting COVID-19. She developed progressive thrombocytopenia, renal failure, and elevated C-reactive protein levels. She was tentatively diagnosed with multisystem inflammatory syndrome in adults (MIS-A) and received steroid pulse therapy. However, she exhibited worsening fluid retention and progressive renal failure, which are not typical of MIS-A. A bone marrow examination showed reticulin myelofibrosis and an increased number of megakaryocytes. Although a definitive diagnosis of TAFRO syndrome was not made according to current diagnostic criteria, we determined that her symptoms were clinically consistent with those of TAFRO syndrome. Combination therapy, including steroid pulse therapy, plasma exchange, rituximab, and cyclosporine, improved her symptoms. There are pathological similarities between hyperinflammation that arises after COVID-19 and TAFRO syndrome in terms of the associated cytokine storms. COVID-19 may have triggered the development of systemic inflammation mimicking TAFRO syndrome in this case.

Biogenesis aberration: One of the mechanisms of thrombocytopenia in COVID-19.

Background: The pathogenesis of COVID-19, including thrombocytopenia, has not been fully clarified. The lungs are a major organ of platelet production and thrombocytopenia induced by severe COVID-19 was proposed. Methods: the change of platelet level was analysed with clinical parameters in 95 hospitalized COVID-19 patients in Wuhan Third Hospital. The production of platelets in the lungs was explored in an ARDS rat model. Results: The level of platelets was negatively correlated with disease severity and was recovered with disease improvement. The non-survivors were accompanied by lower levels of platelet. The odds ratio (OR) of the valley level of the platelet count (PLTlow) was greater than 1, suggesting that PLTlow could be a death exposure factor. The platelet/lymphocyte ratio (PLR) was positively associated with severity of COVID-19, and the platelet/lymphocyte ratio threshold of 248.5 was best correlated with death risk (sensitivity 0.641 and specificity 0.815). To demonstrate the possible biogenesis aberration of platelet in lungs, an LPS-induced ARDS rat model was applied. Lower level of platelet in peripheral and less production of platelet from lungs in ARDS were demonstrated. Though megakaryocyte (MK) number in ARDS lungs is higher than controls, the immature platelet fraction (IPF) in postpulmonary blood is still at the same level as prepulmonary in ARDS rat, indicating that ARDS rats generated fewer platelets in lungs. Conclusion: Our data suggested that COVID-19-induced severe lung inflammation may impair platelet production in the lung. Thrombocytopenia may be mainly caused by platelet consumption for multiorgan thrombosis; however, biogenesis aberration of platelet in the lung induced by diffuse interstitial pulmonary damage cannot be ruled out.

Evaluation of bone marrow aspirates using the automated hematology analyzer Sysmex XN-3000.

This study evaluated the feasibility of the Sysmex XN-3000 automated hematology analyzer for the assessment of total nucleated cells (TNC) and bone marrow (BM) cell density in routine bone marrow aspiration (BMA) samples. A total of 54 BMA samples from 39 hematological patients were evaluated. The number of megakaryocytes was calculated by a specific gating algorithm using the body fluid mode of the WBC differential (WDF) channel. Lipid contents were calculated through a newly developed algorithm utilizing the WDF channel. The ratio of lipid particles over TNCs by the WNR channel was compared with the BM cellularity assessed by the BM biopsy. The myeloid/erythroid (M/E) ratio was calculated by measuring the number of myeloid cells in the WDF channel and the number of nucleated red blood cells (NRBCs) in the WNR channel. XN-3000 counts and microscopic results showed a linear correlation in TNC (R2 =.98, p < .001), megakaryocytes (R2 =.59, p=.002), NRBC (R2 =.84, p < .001), and M/E ratio (R2 =.59, p < .001). There were significant differences in the lipid/TNC ratios of hypercellular, normocellular, and hypocellular BMs measured by XN-3000 (p < .001). Receiver-operating characteristic analysis detected cut-off values of the lipid/TNC ratio of >0.4054 for hypoplasia and <0.157 for hyperplasia. The sensitivity and specificity for hypoplasia were 100% and 88%, and for hyperplasia were 89% and 86%, respectively. XN-3000 provides a quantitative assessment of BM cellularity, supporting the qualitative assessment by myelogram and BM biopsy.

Inhibition of CXCR1/2 reduces the emperipolesis between neutrophils and megakaryocytes in the Gata1low model of myelofibrosis

Emperipolesis between neutrophils and megakaryocytes was first identified by transmission electron microscopy. Although rare under steady-state conditions, its frequency greatly increases in myelofibrosis, the most severe of myeloproliferative neoplasms, in which it is believed to contribute to increasing the transforming growth factor (TGF)-β microenvironmental bioavailability responsible for fibrosis. To date, the challenge of performing studies by transmission electron microscopy has hampered the study of factors that drive the pathological emperipolesis observed in myelofibrosis. We established a user-friendly confocal microscopy method that detects emperipolesis by staining with CD42b, specifically expressed on megakaryocytes, coupled with antibodies that recognize the neutrophils (Ly6b or neutrophil elastase antibody). With such an approach, we first confirmed that the bone marrow from patients with myelofibrosis and from Gata1low mice, a model of myelofibrosis, contains great numbers of neutrophils and megakaryocytes in emperipolesis. Both in patients and Gata1low mice, the emperipolesed megakaryocytes were surrounded by high numbers of neutrophils, suggesting that neutrophil chemotaxis precedes the actual emperipolesis event. Because neutrophil chemotaxis is driven by CXCL1, the murine equivalent of human interleukin 8 that is expressed at high levels by malignant megakaryocytes, we tested the hypothesis that neutrophil/megakaryocyte emperipolesis could be reduced by reparixin, an inhibitor of CXCR1/CXCR2. Indeed, the treatment greatly reduced both neutrophil chemotaxis and their emperipolesis with the megakaryocytes in treated mice. Because treatment with reparixin was previously reported to reduce both TGF-β content and marrow fibrosis, these results identify neutrophil/megakaryocyte emperipolesis as the cellular interaction that links interleukin 8 to TGF-β abnormalities in the pathobiology of marrow fibrosis.

Severe thrombocytopenia in a patient with diffuse toxic goitre

Abstract Introduction Diffuse toxic goitre is an autoimmune disorder with a marked prevalence in women. Thrombocytopenia may coexist with diffuse toxic goitre. Nevertheless, the mechanisms of decline in platelet count alongside with autoimmune thyroid disease have not been extensively investigated. Clinical Case A case of diffuse toxic goitre is reported in a 55-year-old man who has been receiving continuous treatment with methimazole for 7 years. The physical examination revealed an asthenic man, with an enlarged thyroid gland, inactive bilateral orbitopathy and multiple ecchymosis, but no lymphadenopathy or hepatosplenomegaly. Laboratory analysis revealed suppressed thyroid stimulating hormone (TSH) 0.01 mlU/ml, normal free T4 level 21.60 pmol/l, increased free T3 6.99 pmol/l and thyroid stimulating immunoglobulin (TSI) level of 23.60 IU/L. Complete blood count revealed severely decreased platelets 3×10^9 / l, decreased hemoglobin 110.00 g/l and hematocrit 21.90%, normal leucocytes 9.58×10^9/l, low prothrombin Index 11% and International normalized ratio (INR) 7.00. Additional serological investigations excluded HIV infection and C viral hepatitis. The bone marrow aspiration disclosed a rich cellular and polymorph marrow, an increased number of megakaryocytes suggesting that the thrombocytopenia is due to platelet destruction. The patient has recovered from viral pneumonia caused by SARS-COV-2, 1 month prior to the medical consultation. He received platelet transfusion and was started on 1 mg/kg oral prednisolone followed by a gradual taper and improvement of platelet count. Thrombocytopenia observed in diffuse toxic goitre may be mediated by both metabolic and immunological phenomena. In our case, the following causes may be considered: (1) an overlapping autoimmune process; (2) methimazole creating destructive antibodies; (3) COVID-19 disease. An autoimmune process is capable of triggering both conditions by activation of the reticuloendothelial system by thyroid hormones along with a cross-reaction between thyroid antibodies and platelet epitopes. Methimazole generates a drug-dependent immune response against platelets, involving platelet endothelial cell adhesion molecules and thus provoking thrombocytopenia. SARS-CoV-2 can induce thrombocytopenia by increasing the number of autoantibodies and immune complexes that destroy platelets. Lung damage by decreasing pulmonary capillary bed and platelet aggregation in the lungs indirectly leads to a reduced number of circulatory platelets. Conclusion Severe thrombocytopenia was detected in a patient with Grave's disease, in whom the number of platelets was 50 times below the lower limit of the reference range. Coexistence of thrombocytopenia may be explained by (1) an overlapping autoimmune process; (2) the effect of methimazole; (3) its occurrence or aggravation secondary to COVID-19.

Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

Concurrent Occurrence of Nasopharyngeal and Disseminated Cutaneous Rhinosporidiosis: A Rare Presentation.

Concurrent Occurrence of Nasopharyngeal and Disseminated Cutaneous Rhinosporidiosis: A Rare Presentation.

Histamine and Serotonin Levels in Bone Marrow Stem Cells Niche as Potential Biomarkers of Systemic Mastocytosis and Myeloproliferative Disorders

Bone marrow studies currently provide a lot of valuable information in the diagnostics of hematological diseases including hematopoietic stem cells disorders. Our studies on low-molecular weight organic compounds in bone marrow stem cell niche in various pathogenic conditions, revealed relatively high variability of histamine levels in different groups of hematological diseases. It was also found that serotonin levels were significantly lower than those typically measured in peripheral blood as well as many have the influence on stem cells proliferative potential. This paper presents findings from quantitative and statistical analyses of histamine and serotonin levels. Bone marrow collected from patients undergoing routine diagnostic procedures for hematological diseases and receiving inpatient treatment were analyzed. Histamine and serotonin levels were measured using hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry. Obtained data were analyzed statistically and correlated with the diagnosed groups of hematological diseases and the parameters of complete blood counts. Histamine was found in all tested samples, including those from patients without malignancy, and the reported levels were comparable to the reference values in blood. This observation allows us to assume that bone marrow cells can produce and accumulate histamine. Moreover, the statistical analysis revealed a significant relationship between histamine levels and diagnosed mastocytosis, and between histamine levels and myeloproliferative neoplasms. Different results were obtained for serotonin, and its concentrations in most cases were below the limit of quantification of the method used (< 0.2 ng/mL), which can only be compared to peripheral blood plasma. In a few cases, significantly higher serotonin levels were observed and it concerned diseases associated with an increased number of megakaryocytes in the bone marrow.

Dual specificity tyrosine phosphorylation regulated kinase 1B inhibition promotes megakaryocyte polyploidization and platelet production.

Platelets are produced from mature megakaryocytes which undergo polyploidization and proplatelet formation. Cell cycle regulation plays a crucial role in megakaryocyte terminal differentiation especially in polyploidization. Dual specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) controls cell cycle progression in cancer cells. The objective of this study was to determine DYRK1B function in megakaryocyte maturation and platelet production. A DYRK1B knock out mouse (DYKO) was generated with increased peripheral platelet count compared to the wild type (WT) mouse without affecting megakaryocyte numbers in bone marrow. Polyploidy and proplatelet formation were significantly enhanced when DYRK1B was depleted in vitro. DYRK1B inhibition promoted megakaryocyte maturation by simultaneously upregulating cyclin D1 and downregulating P27. Furthermore, there was platelet restoration in two mice disease models of transient thrombocytopenia. In summary, DYRK1B plays an important role in megakaryocyte maturation and platelet production by interacting with cyclin D1 and P27. DYRK1B inhibition has potential therapeutic value in transient thrombocytopenia treatment.