Emperipolesis of hematopoietic cells in myelocytic leukemia. Electron microscopic and phase contrast microscopic studies

In case of blast crisis of chronic myelocytic leukemia, the blast cells contained several kinds of normal hematopoietic cells. The peroxidase reaction was strongly positive in the neutrophilic granules of the engulfed neutrophils. These engulfed cells appeared to be normal and the limiting membranes of the engulfing cells seemed to be intact. We speculated therefore that this phenomenon might be emperipolesis. In a case of chronic myelocytic leukemia and a case of acute myelocytic leukemia, some megakaryoblasts showed the same phenomenon. These megakaryoblasts did not phagocytize latex particles. The limiting membranes of the engulfing megakaryoblasts were stained with ruthenium red but those of the engulfed hematopoietic cells were not stained. By phase microscopy, the engulfed cells were actively moving inside the megakaryoblasts and it was observed that the engulfed cells were actually living within the engulfing cells. These results demonstrated that this phenomenon was emperipolesis. Observations with an electron microscope and the phase microscope are indispensable for distinguishing emperipolesis from phagocytosis.     

Megakaryocytes emperipolesis in bone marrow of the patients with non-Hodgkin's lymphoma

Emperipolesis of hematopoietic cells within the cytoplasm of the megakaryocytes was most often described in association with various pathologic conditions. The aim of the research was estimation of the incidence of emperipolesis in the bone marrow of the patients with non-Hodgkin's lymphoma (NHL). 30 patients with different histological types of NHL (chronic lymphocytic leukaemia--CLL, hairy cell leukaemia--HCL, multiple myeloma--MM) in compliance with clinical stage of the disease, patient's age and sex were analyzed. Trephine biopsies of the bone marrow were carried out in fixative solution and paraffin embedding. Hematoxylin and eosin and monoclonal antibody CD 61 were applied on thin sections. Phenomenon of megakaryocytic emperipolesis in human bone marrow was found in 6 cases: in 5 cases in CLL and in 1 case in HCL. In most of them emperipolesis was related to single megakaryocytes. We observed in the cytoplasm of the megakaryocytes single hematopietic cells-most often lymphocytes, rarely eosinophilic granulocytes. We found no correlations between histological types of NHL, clinical stage of the disease, patients' age, sex and the incidence of megakaryocytic emperipolesis.     

Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias

Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar rat bone marrow was fixed and processed for transmission electron microscopy after VCR administration alone, after 5-fluorouracil (5-FU) administration alone, or after 5-FU followed by intravenous injection of 0.1–1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (～two-third of megakaryocytes) at VCR dosages of 0.75–1.0 mg/kg. The majorityof megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted α-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstrate these abnormalities, with the exception of an increase in dense compartments. Platelets from rats treated with VCR aloene or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar rats as well as untreated Wistar Furth rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte α-granules. © 1995 Wiley-Liss, Inc.     

Modulation of megakaryocyte emperipolesis by phlebotomy: megakaryocytes as a component of marrow-blood barrier

The presence of marrow cells within the cytoplasm of megakaryocytes has been documented and is attributed to the phenomenon of emperipolesis. In a previous study most patients demonstrating this phenomenon had or were suspected of having blood loss. To find out if these are causally related, we induced acute and chronic blood loss in rats and quantitatively measured the emperipolesis index (EI). EI dropped after acute blood loss but rose after chronic blood letting, indicating that blood loss can modulate this phenomenon. In the marrow, megakaryocytes are located preferentially on the abluminal side of sinus endothelium and it is postulated that in the state of heightened demand for cell delivery from marrow into the circulation, some cells take a transmegakaryocytic route to enter the circulation. This concept incorporates megakaryocytes as a component of the marrow-blood barrier.     

Lectin histochemistry of human bone marrow: investigation of trephine biopsy specimens in normal and reactive states and neoplastic disorders

Summary The lectin binding pattern of bone marrow cells in normal and reactive states and in various neoplastic disorders was investigated using trephine biopsy specimens taken from the iliac crest. The tissue samples were routinely processed (fixed in formalin and embedded in paraffin wax) and subjected to mild decalcification with EDTA. The following results were obtained. (1) More than half of the 23 fluoresceinated lectins used reacted with normal blood cells and/or their neoplastic derivatives. Inhibition tests with the appropriate sugars confirmed the specificity of binding for the majority, but not all, of the lectins. (2) WGA, Con A, PSA, STA and RCA₆₀ and RCA₁₂₀ produced a particularly intense reaction with normal, reactive and neoplastic myeloid cells. Erythroblasts exhibited weak staining in a few cases by a few lectins (WGA producing the strongest staining), while megakaryocytes nearly always remained unstained. Neoplastic lymphoid cells in various lymphoproliferative disorders and plasmacytoma cells generally reacted with the same lectins as the myeloid cells. (3) Since neoplastic myeloid cells in various myelodysplastic and myeloproliferative disorders exhibited a lectin binding pattern similar to that of myeloid cells in normal and reactive bone marrow, it is unlikely that lectin histochemistry of the bone marrow will prove of great value in the diagnosis of myelodysplastic—myeloproliferative disorders.     

Megakaryocytes in a hemorrhagic pleural effusion caused by anticoagulant overdose

Sudden widespread hemorrhage, including a hemorrhagic pleural effusion, developed in a patient due to an overdose of anticoagulant. The pleural fluid contained megakaryocytes. Necropsy did not reveal myeloid metaplasia or any myeloproliferative disorder. Since megakaryocytes are normally found in pulmonary parenchyma, their presence in the pleural fluid was attributed to the hemorrhagic condition of the lung, which enabled megakaryocyte-containing blood to enter the pleural cavity.     

Notch signaling specifies megakaryocyte development from hematopoietic stem cells

In the hematopoietic system, Notch signaling specifies T cell lineage fate, in part through negative regulation of B cell and myeloid lineage development. However, we unexpectedly observed the development of megakaryocytes when using heterotypic cocultures of hematopoietic stem cells with OP9 cells expressing Delta-like1, but not with parental OP9 cells. This effect was abrogated by inhibition of Notch signaling either with gamma-secretase inhibitors or by expression of the dominant-negative Mastermind-like1. The importance of Notch signaling for megakaryopoietic development in vivo was confirmed by using mutant alleles that either activate or inhibit Notch signaling. These findings indicate that Notch is a positive regulator of megakaryopoiesis and plays a more complex role in cell-fate decisions among myeloid progenitors than previously appreciated.     

Hematologic disease induced in BALB/c mice by a bcr-abl retrovirus is influenced by the infection conditions.

Irradiated mice reconstituted with bone marrow cells infected with a retrovirus carrying the bcr-abl oncogene of human chronic myeloid leukemia are subject to a range of neoplastic hematopoietic diseases, both myeloid and lymphoid. Comparison of DBA/2 and C57BL/6 mice has revealed a marked strain difference in susceptibility to the various tumor types. The present study, performed with BALB/c mice, indicates that the kinetics and nature of the induced disease can be modulated by the infection procedure, as well as the genetic background, and that retroviral regulatory sequences may influence the outcome. A distinctive clonal myeloproliferative disorder, somewhat akin to chronic myeloid leukemia but with prominent erythroid and mast cell components, as well as granulocytic excess, was characterized.     

Human EVI9, a homologue of the mouse myeloid leukemia gene, is expressed in the hematopoietic progenitors and down-regulated during myeloid differentiation of HL60 cells

Evi9, a common site of retroviral integration in BXH2 murine myeloid leukemias, encodes a C2H2 zinc finger protein and is overexpressed in these leukemic cells. To investigate a possible role of EVI9 in the human hematopoietic system, we isolated the cDNA clone of the human homologue. Human EVI9, located on the chromosome 2p13 region, contains an open reading frame of 797 amino acids that is 98.7% identical to the mouse protein. RT-PCR analysis of purified human hematopoietic cells showed that EVI9 is expressed in CD34-positive myeloid precursors, B cells, monocytes, and megakaryocytes, but only weakly in T lymphocytes, suggesting that EVI9 may play an important role in hematopoiesis. Furthermore, EVI9 was down-regulated during myeloid differentiation of HL60 cells induced by all-trans-retinoic acid, whereas the expression remained during monocytic differentiation induced by phorbol 12-myristate 13-acetate. These results indicate a distinct role for EVI9 in human hematopoietic cells and suggest that EVI9 may cause leukemia through inhibition of myeloid differentiation.     

Emperipolesis of lymphoid cells by human follicular dendritic cells in vitro.

Isolated follicular dendritic cells (FDCs) showed true and pseudoemperipolesis of fresh tonsillar lymphocytes, even after long-term (50-day) cultivation. Emperipolesis by FDCs was not restricted by allotype specificity, nor was it inhibited by the addition of antibodies against MHC-I & II antigens. Follicular dendritic cells predominantly engulfed B-cells; monocytes and macrophages were not found between FDC cytoplasmic extensions. When highly purified T-cell populations were added to FDC cultures emperipolesis of T-cells occurred, particularly those of the CD4-positive phenotype. Mitoses appeared within 6 h in the emperipolesed lymphocytes and, after an additional 18 h, some lymphocytes exhibited apoptosis.     

Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.     

Transient myeloproliferative disorder in Down syndrome presenting with ascites: a case report

BACKGROUND: An increased frequency of acute myelogenous leukemia is a well known feature in children with Down syndrome. In addition, transient myeloproliferative disorders (TMD), which may mimic acute leukemia, also occur in neonates with Down syndrome. TMD is recognized shortly after birth or in the neonatal period and is characterized by leukocytosis and thrombocytopenia, which resolve spontaneously in four to six weeks. CASE: A 1.5-month-old, male infant born with Down syndrome and patent ductus arteriosus presented with abdominal distention due to ascites. Cytology of the fluid revealed immature myeloid cells and megakaryocytes. Flow cytometry of the ascitic fluid confirmed the presence of immature myelomonocytic cells. A complete hematologic evaluation along with the clinical findings supported the diagnosis of TMD in Down syndrome. CONCLUSION: TMD is an uncommon syndrome strongly associated with Down syndrome. Since the abnormal laboratory findings are seen primarily in the peripheral blood, it is usually diagnosed by a hematopathologist without much difficulty. Our case demonstrates the importance of cytopathologist familiarity with this entity so as not to erroneously diagnose a leukemic process. This is extremely important since most cases of TMD spontaneously resolve within a few weeks to months and do not require treatment other than supportive measures.     

Mapping of MN1 Sequences Necessary for Myeloid Transformation

The MN1 oncogene is deregulated in human acute myeloid leukemia and its overexpression induces proliferation and represses myeloid differentiation of primitive human and mouse hematopoietic cells, leading to myeloid leukemia in mouse models. To delineate the sequences within MN1 necessary for MN1-induced leukemia, we tested the transforming capacity of in-frame deletion mutants, using retroviral transduction of mouse bone marrow. We found that integrity of the regions between amino acids 12 to 458 and 1119 to 1273 are required for MN1’s in vivo transforming activity, generating myeloid leukemia with some mutants also producing T-cell lympho-leukemia and megakaryocytic leukemia. Although both full length MN1 and a mutant that lacks the residues between 12–228 (Δ12–228 mutant) repressed myeloid differentiation and increased myeloproliferative activity in vitro, the mutant lost its transforming activity in vivo. Both MN1 and Δ12–228 increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparisons of purified MN1-CMP and Δ12–228-CMP cells showed many differentially expressed genes including Hoxa9, Meis1, Myb, Runx2, Cebpa, Cebpb and Cebpd. This collection of immediate MN1-responsive candidate genes distinguishes the leukemic activity from the in vitro myeloproliferative capacity of this oncoprotein.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.