Bone marrow response to acute and chronic Trypanosoma congolense infection in multimammate rats (Mastomys coucha)

The femoral bone marrow of multimammate rats (n=90), aged 3-8 weeks, experimentally infected with different doses of Trypanosoma congolense was examined by light and electron microscopy. Some animals died from trypanosomosis, but groups of 10 were killed at 4-8, 9-16, 20-24, 30, 40, 50 and 60 days post-infection (dpi). In the acute stage of infection (4-8 dpi) the bone marrow invariably showed a striking increase in erythropoiesis, characterized by an increase in the number of mitotic figures and erythroblastic islands and by a marked decrease in the myeloid:erythroid cell ratio. Later in the infection, erythropoietic activity decreased, while erythrophagocytosis, granulopoiesis, megakaryopoiesis and plasma cell population increased. In chronic infection (16-60 dpi), erythropoietic activity decreased, while intra- and extra-vascular erythrophagocytosis greatly increased. There was also an increase in the bone marrow stroma cells. Excessive erythrophagocytosis by these cells led to the formation of myelin figures and cytoplasmic telephagolysosomes. Degeneration and necrosis of neutrophils lining the adluminal surfaces of the blood sinuses were observed. It is concluded that in the acute stage of the infection, the bone marrow is responsive to the anaemia and that in the chronic stage, dyserythropoiesis and increased erythrophagocytosis by the expanded and activated cells of the mononuclear phagocytic system play an important role in the production of anaemia. Copyright Harcourt Publishers Ltd.     

ELECTRON MICROSCOPIC STUDIES OF THE SPLEEN AND LIVER IN HEREDITARY SPHEROCYTOSIS

Electron microscopic findings of the red pulp of the spleen and liver from three patients with hereditary spherocytosis have been reported.
The sinus lumen of the spleen contained various amounts of red cell with reduced deformability. Pronounced engorgement of erythrocytes in the cordal space and various stages of erythrophagocytosis by the cordal macrophages were characteristic findings. The sinus lining cells also showed erythrophagocytosis and contained numerous dense bodies. Cordal macrophages and fibrous elements seemed to be more increased in the older patient.
Acid phosphatase activity of varied intensity was demonstrated in the erythrophagocytic vacuoles of cordal macrophages. As the intracellular degradation progressed, more intense activity of this enzyme was noted, indicating their lysosomal origin.
Splenic conditioning and enhanced destruction of defective red cells in the spleen were the main cause of hemolysis in hereditary spherocytosis.
In the liver, erythrophagocytosis by the Kupffer cells was also not infrequently noted, but the liver was estimated to play a minor role in HS.
The mechanism of hemolysis and the role of the spleen and liver in elimination of hereditary spherocytes have been discussed.     

AN AUTOPSY CASE OF AUTOIMMUNE HEMOLYTIC ANEMIA

An autopsy case of autoimmune hemolytic anemia which revealed typical clinical symptoms, and complicated with toxic epidermal necroly-sis in its terminal stage, has been reported.
Morphological changes which imply the pathogenesis of this disease have been reported. The liver was the main site of extravascular hemolysis. Various stage of red cell destruction in the Kupffer cells, erythrophagocytosis by the mononuclear cells in the peripheral blood and multiple thrombi might be relatively specific findings which suggest the hemolytic process of this disease. Hemosiderosis, erythroid hyperplasia in the bone marrow and myeloid metaplasia were also noted.     

Primary histiocytic sarcoma of the spleen associated with erythrophagocytic histiocytosis

We report an exceptional case of a histiocytic sarcoma presenting as a primary isolated spleen tumor in a 71-year-old woman. The neoplastic cells in the cords and sinuses of the red pulp formed multiple lobulated tumors, which were detected in vivo by ultrasound scan. The medium cells, large cells and the giant cells expressed CD68, a histiocyte-associated marker, lysozyme and S100 protein. All these cells were negative for B- and T-cell markers, cytokeratins, melanosome markers (HMB45) and CD1a (Langerhans' cells). Many tumor cells displayed strong erythrophagocytosis and sometimes lymphocytophagocytosis. In addition, numerous histiocytes with morphology indistinguishable from reactive macrophages also exhibited a strong erythrophagocytosis, and were found in the tumor as well as in the normal splenic parenchyma. Despite multi-agent chemotherapy, the patient suffered from a relapse in the liver, with a rapid fatal outcome. A literature review showed that such a primary splenic presentation with multiple tumors is rare. In contrast, in systemic malignant histiocytosis, secondary spleen involvement occurs more frequently but with diffuse infiltration. The association with a reactive histiocytosis with erythrophagocytosis corresponds to "histiocytic medullary reticulosis", as previously described by Scott and Robb-Smith.     

A fluorometric quantitative erythrophagocytosis assay using human THP-1 monocytic cells and PKH26-labelled red blood cells

Removal of senescent, damaged or diseased red blood cells (RBCs) from the circulation in vivo occurs by a process known as erythrophagocytosis. The exact details of the signaling mechanisms that mark RBCs for recognition and influence erythrophagocytosis are still not completely understood. The aim of this study was to develop a quantitative, fluorometric erythrophagocytosis assay for human RBCs and phagocytes to aid elucidation of the biological mechanisms regulating erythrophagocytosis. RBCs were labelled with the lipophilic fluorescent dye PKH26 and incubated with the human monocytic cell line THP-1 at 37 degrees C for 45 min. Non-phagocytosed RBCs were lysed with hypotonic saline. Phagocytosed PKH26-labelled RBCs within THP-1 cells were detected with a fluorescence plate-reader and quantitated using a standard curve of known numbers of PKH26-labelled RBCs. Assay conditions were optimised for the numbers of phagocytes and RBCs, incubation time and fluorescence excitation and emission wavelengths. Erythrophagocytosis was also assessed by flow cytometry to determine the proportion of THP-1 cells with ingested RBCs and showed good correlation (P=0.7) between the two methods. The quantitative, fluorometric plate assay is very sensitive and has good reproducibility, making it a useful tool to investigate the biological mechanisms that regulate erythrophagocytosis of normal and diseased RBCs.     

An acute myeloproliferative disorder characterized by myelofibrosis and blast cells that express phenotypic properties associated with multiple hematopoietic lineages

The authors investigated the blast cells obtained from two patients with acute myelofibrosis with the use of recently developed immunochemical and ultrastructural markers. They intended to examine the expression of megakaryoblastic, erythroblastic, myeloblastic, and monoblastic properties in these cells. Respectively, 20% and 15% of the blasts from patient 1 and patient 2 expressed a megakaryoblastic phenotype as determined by reactivity with a polyclonal platelet glycoprotein antisera (PGP). However, 55% and 73% of these patients' blasts also exhibited myeloid/monocytic properties. Ultrastructural studies clearly demonstrated findings consistent with the presence of myeloblasts, monoblasts, and erythroblasts, as well as undifferentiated agranular blasts. These findings demonstrate the existence of a disorder in which blast cells simultaneously express properties associated with multiple hematopoietic lineages. This disorder is characterized by impaired terminal differentiation.     

Acute myeloblastic leukemia with extensive erythrophagocytosis mimicking malignant histiocytosis

The authors report a case of acute myeloblastic leukemia in which erythrophagocytosis by the leukemic cells was so extensive as to mimic malignant histiocytosis at postmortem. It is postulated that premature expression of phagocytic function in leukemic cells and disseminated intravascular coagulopathy, which accompanied the initial clinical presentation, explained the unusually prominent erythrophagocytosis.     

Erythrophagocytosis by the sinus endothelial cell of the spleen in haemolytic anaemias

Summary An ultrastructural study of spleens from patients with a heterogeneous group of haemolytic anaemias was undertaken in order to determine whether sinus endothelial cells have erythrophagocytic ability. In most cases, sinus endothelial cells contained erythrocytes and various stages of intracellular degradation of engulfed erythrocytes were noted. Though the frequency of erythrophagocytosis varied from case to case, phagocytosis of erythrocytes by the endothelial cell was more frequent in cases in which cordal macrophages showed active erythrophagocytosis. These results suggest that the sinus endothelial cells have erythrophagocytic ability in certain pathological states, especially when the demands for the removal of defective erythrocytes are increased. However, the bulk of erythrophagocytosis is carried out by cordal macrophages, and endothelial phagocytosis has a minor significance in the development of haemolytic anaemia. Two possible processes by which erythrocytes come into contact with sinus endothelial cells are suggested.     

Erythrophagocytosis in sickle cell anemia: statistical evidence for a biological phenomenon

The precise role of erythrophagocytosis in sickle cell disease is not known. Using hematological data from three studies and 791 subjects comprising of eight epidemiological groups, we found a strong statistical support for the hypothesis that erythrophagocytosis is increased in sickle cell trait, that neutrophils and lymphocytes are the most likely cells involved in erythrophagocytosis in these subjects and that increased erythrophagocytosis may for a mechanistic explanation for an increased risk of vaso-occlusive crisis in sickle cell trait. Statistically, erythrophagocytosis was not increased in subjects with homozygous sickle cell disease. Our findings offer an interesting mechanistic implication about the presence of a strong autoimmune component of sickle cell trait that can be explained by the well recognized interplay between the receptor molecule signal regulatory protein-alpha (SIRP-alpha) on the phagocyte and its ligand, CD47, on the red blood cell. Our findings also support further and closer evaluation of the other hypothesized mechanisms by which neutrophils and lymphocytes partake in differential degree of erythrophagocytosis in subjects heterozygous for the sickle hemoglobin. Finally, translation of these findings into a clinical realm suggests that the extent of erythrophagocytosis, as measured by peripheral blood hematological indicators, can serve as an important indicator of the likelihood of future vaso-occlusive crisis events in subjects of sickle cell trait.     

Spheroidal filamentous inclusion body cells in von Recklinghausen's disease

Summary Cells with spheroidal filamentous cytoplasmic bodies, distinctive by both light and electron microscopy, were found in a neoplasm arising from the sciatic nerve of a patient with von Recklinghausen's disease. Tissue fixed with formalin and embedded in paraffin for three years was deparaffinized, reprocessed, and examined with the electron microscope. The morphology of the spheroidal body cells, the close resemblance to erythrophagocytosis, and the possible significance of the changes are discussed.     

p53 expression in myeloid cells of myelodysplastic syndromes. Association with evolution of overt leukemia.

To assess p53 expression in the hematopoietic cells of the bone marrow in premalignant as well as malignant conditions, we examined immunohistochemically bone marrow biopsies from patients with myelodysplastic syndromes (MDS, n = 51), acute myeloid leukemia (n = 42) and as a nonneoplastic condition, aplastic anemia (n = 20) and samples from individuals who had no hematological disorder (control, n = 12). Nuclear accumulation of p53 protein was found in seven of 51 patients with MDS (14%) and two of 42 acute myeloid leukemia patients (5%), whereas patients with aplastic anemia and control subjects were uniformly negative for p53 protein. In the bone marrow of patient with MDS, p53-positive cells constituted about 5 to 30% of the total bone marrow cells. Two-color immunohistochemical analysis revealed that the p53-positive cells were also positive for the myeloid cell marker. Half of the MDS cases that evolved to overt leukemia (seven of 14) exhibited positive p53 reaction in the bone marrow at the time of initial diagnosis. This frequency (50%) was significantly higher than that in de novo acute myeloid leukemia cases. All of the seven MDS cases that exhibited p53 expression at the time of initial diagnosis developed overt leukemia later, and p53 expression was maintained throughout the progression of MDS. The results suggest that p53 mutations that occur in the myeloid cells in MDS may confer a growth advantage to these cells resulting in the progression to overt leukemia. Thus, immunohistochemical examination for p53 is very useful for predicting the evolution to overt leukemia from MDS.     

Clonality of chronic neutrophilic leukaemia associated with myeloma: analysis using the X-linked probe M27 beta.

AIMS--To determine whether myeloid proliferation was monoclonal or polyclonal in a woman with chronic neutrophilic leukaemia and myeloma. METHODS--The X-linked probe, M27 beta was used to determine the clonality of the neutrophil population by analysis of restriction fragment length polymorphisms and X inactivation pattern. RESULTS--A polyclonal pattern of X inactivation was obtained for the neutrophil population in this patient. CONCLUSION--The myeloid expansion in chronic neutrophilic leukaemia associated with myeloma represents a polyclonal reactive response to the plasma cell clone rather than a co-existent myeloproliferative disorder.     

BACH1 regulates erythrophagocytosis and iron-recycling in β-thalassemia

Macrophages play essential roles in erythrophagocytosis and iron recycling. β-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of β-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in β-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with β-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.     

Malignant histiocytosis: a case report of a rare tumour presenting with spontaneous splenic rupture

Malignant histiocytosis is a rare invasive proliferation of neoplastic histiocytes. Cases previously reported as malignant histiocytosis were shown to be lymphomas of T or B lineage, especially anaplastic large-cell lymphomas. A case of malignant histiocytosis is described, in which a patient presenting with symptoms suggestive of pneumonia suddenly deteriorated and died. At autopsy, a large quantity of fresh blood, originating from several ruptured nodules on the enlarged spleen, was seen in the peritoneal cavity. Extensive infiltration by pleomorphic tumour cells and erythrophagocytosis by tumour cells were seen on histological examination of the spleen. Immunohistochemical analysis and staining were carried out. This is the second reported case of malignant histiocytosis presenting with spontaneous splenic rupture.     

Cytomorphology of familial hemophagocytic syndrome

Familial hemophagocytic syndrome (FHS) is a rare, fatal disorder of childhood demonstrating failure to thrive, fever, hepatosplenomegaly (HSM), recurrent infections, pancytopenia, and histologically, the infiltration of reticuloendothelial organs by benign-appearing histiocytes demonstrating hemophagocytosis. We report two fatal cases of FHS including a 3 year-old female who underwent fine-needle aspiration (FNA) biopsy of the liver in the initial workup of the disease (case 1) and an 8 month-old boy with ascites and HSM having peritoneal fluid cytology submitted as the first specimen for morphologic examination (case 2). In case 1, the FNA cytologic findings included benign hepatocytes and scattered mature and reactive lymphocytes and histiocytes. The histiocytes demonstrated fine to coarse cytoplasmic vacuoles and erythrophagocytosis. The diagnosis was confirmed at autopsy which revealed extensive lymphohistiocytic infiltrates in various organs including the central nervous system. In case 2, the peritoneal fluid cytology specimen contained numerous atypical and degenerating mononuclear lymphoreticular cells which were dispersed as a single cell suspension admixed with infrequent mesothelial elements; hemophagocytosis was not appreciated. Subsequent liver biopsy revealed portal tracts and sinusoids infiltrated by benign but atypical histiocytes with hemophagocytosis. Bone marrow examination and then autopsy confirmed the diagnosis of FHS. A panel of immunocytochemical studies was performed in the first case which was an aid in confirming the diagnosis of FHS and ultrastructural examination of the second case revealed well-developed erythrophagocytosis. Both patients had siblings who died of FHS. Although not diagnostic, cytomorphology may suggest FHS.     

Animal model of fatal human monocytotropic ehrlichiosis

Human monocytotropic ehrlichiosis caused by Ehrlichia chaffeensis is a life-threatening, tick-borne, emerging infectious disease for which no satisfactory animal model has been developed. Strain HF565, an ehrlichial organism closely related to E. chaffeensis isolated from Ixodes ovatus ticks in Japan, causes fatal infection of mice. C57BL/6 mice became ill on day 7 after inoculation and died on day 9. The liver revealed confluent necrosis, ballooning cell injury, apoptosis, poorly formed granulomas, Kupffer cell hyperplasia, erythrophagocytosis, and microvesicular fatty metamorphosis. The other significant histological findings consisted of marked expansion of the marginal zone and infiltration of the red pulp of the spleen by macrophages, interstitial pneumonitis, and increased numbers of immature myeloid cells and areas of necrosis in the bone marrow. Ehrlichiae were detected by immunohistology and electron microscopy in the liver, lungs, and spleen. The main target cells were macrophages, including Kupffer cells, hepatocytes, and endothelial cells. Apoptosis was detected in Kupffer cells, hepatocytes, and macrophages in the lungs and spleen. This tropism for macrophages and the pathological lesions closely resemble those of human monocytotropic ehrlichiosis for which it is a promising model for investigation of immunity and pathogenesis.     

Common reactive erythrophagocytosis in axillary lymph nodes

Erythrophagocytosis by histiocytes in the sinuses of axillary lymph nodes is a common yet little-known phenomenon. The axillary lymph node dissections of 23 patients were studied by light microscopy and graded for the amount of erythrophagocytosis. None of the patients had evidence of a systemic hemolytic process. Nineteen of them exhibited some degree of erythrophagocytosis, and this was present even in four of the six patients who never had a prior breast biopsy. Breast biopsy was associated with massive degrees of erythrophagocytosis in 8 of 17 patients, and after a postbiopsy interval of two weeks 11 of 13 patients had hemosiderin deposition in the lymph nodes, evidence of red blood cell breakdown. This study serves to substantiate statements, made by others without confirmatory data, that large degrees of erythrophagocytosis may be seen in axillary lymph nodes after breast biopsies and that small amounts of erythrophagocytosis may be identified in lymph nodes of patients with no prior trauma history.     

Reciprocal translocation (3;5)(q26;q22) and possible BCHE gene involvement in an unusual myelogenous disorder with both myeloproliferative and dysplastic features

We report on a 77-year-old male patient who presented with an unusual myelogenous disorder exhibiting both myeloproliferative and dysplastic features. The patient suffered from leukocytosis, eosinophilia, basophilia, transfusion dependent anemia, and rapidly progressing thrombocytopenia. Classical chromosome analysis and fluorescence in situ hybridization (FISH) revealed a reciprocal t(3;5)(q26;q22). Using yeast artificial chromosome (YAC) probes, the breakpoint on chromosome 3 was localized to the butyrylcholinesterase (BCHE) gene (3q26.1-q26.2). This gene has recently been implicated in the regulation of myeloid cells. Whether the BCHE gene was also involved in the deregulation of myelopoiesis, causing the unusual clinical picture in this case, remains unknown.     

Ultrastructure of acute myeloid leukemia arising in multiple myeloma

Bone marrow from a case of multiple myeloma in which acute myeloid leukemia supervened four years after diagnosis was examined with the electron microscope. Two distinct populations of neoplastic cells, one plasmacytoid and the other myeloid, were identified in the marrow. It is concluded that the acute leukemia that developed in this patient was a distinctly new neoplasm arising from the myeloid series of cells. Since in nearly all previously reported cases, and in our patient, alkylating agents had been administered, it is thought that this may have been a factor in the development of acute myeloid leukemia.