Malformation of the endoplasmic reticulum system evolving into giant inclusions and Auer bodies in acute promyelocytic leukemia: an ultrastructural study of 6 cases.

The endoplasmic reticulum（ER）is the largest membranous network serving as a region for protein, lipid and steroid synthesis, transport and storage. Detailed information about ER-cisternae, ER-tubules and rough endoplasmic reticulum (rER) is scarce in human blood cells. This study describes a series of giant inclusions and Auer bodies in promyeloblasts in six patients with acute promyelocytic leukemia (APL), by light microscopy, transmission electron microscopy (TEM) and cytochemical stains. TEM revealed that giant inclusions and pro-Auer bodies were associated with rER and surrounded by tubular structures composed of degenerated or redundant membrane in promyeloblasts, which corresponded with elements of the ER system. This paper reveals that in the promyeloblasts of APL, ER is the source of and transforms progressively into giant inclusions and Auer bodies.

Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes syndrome: a case report.

A biopsy of gastrocnemius muscle from a patient with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome was studied histologically in semithin sections stained by hematoxylin-and-eosin (H&E) and toluidine blue, and ultrathin sections by transmission electron microscopy (TEM). H&E stain demonstrated typical ragged-red fibers (RRFs) and affected fibers in fascicles. Toluidine-blue stain showed an irregular meshwork in the center of RRFs. TEM demonstrated damaged myofibrils and variations in mitochondrial structure in RRFs and affected fibers. Dense mitochondria were compacted with cristae and pleomorphic electron-dense inclusions. Lucent mitochondria included paracrystalline inclusions with a parking lot appearance. At high magnification, the paracrystalline inclusions were composed of plates that paralleled and connected with mitochondrial cristae. These observations indicated that electron-dense granular and paracrystalline inclusions resulted from cristal degeneration and overlapping in mitochondria in MELAS syndrome.

Foam cell origination from degenerated vascular smooth muscle cells in atherosclerosis: An ultrastructural study on hyperlipidemic rabbits.

To clarify foam cell origination in atherosclerosis, a series of morphologic and ultrastructural alterations of vascular smooth muscle cells (VSMCs) and foam cells were studied by light and electron microscopy in atherosclerotic aortas from hyperlipidemic rabbits induced for 5 weeks. The study exhibited that VSMCs were severely degenerated and damaged, including irregular shapes, expanded mitochondria, aplenty lipid droplets, and disarranged myofilaments in cytoplasm in media adjacent to atheromatic bottoms. Most lipid laden cells shared interphase structures of VSMCs and foam cells, and some dissolved spindle cells contained lipid droplets, lipofuscin, and rod-like CCs in cytoplasm also. The result demonstrated that VSMCs were degenerated and transformed into foam cells in atherosclerosis, which was responsible for the accumulation of lipid and cholesterol crystals in atherosclerotic arteries.

One cell one niche: hematopoietic microenvironments constructed by bone marrow stromal cells with fibroblastic and histiocytic features.

Hematopoietic microenvironments have been extensively studied, especially focusing on regulation of hematopoietic stem cells (HSCs) in HSC niche following progress of molecular biology in resent years. Based on prior morphological achievements from 1970s, the characteristics of cellular compartments and bone marrow stromal cells (BMSCs) were studied ultrastructurally in human and mice bone marrow in the present study. The samples, human bone marrow granules, were collected from bone marrow aspirations (BMAs) of 20 patients with hematocytopenia and isolated BMSCs were found undesignedly in nucleated cells of BMAs of the patients. Femoral bone marrow samples were collected from 6-week-old three sacrificed mice. Detailed images illustrated maturing hematopoietic cells harbored individually in honeycomb-like microenvironment constituted by BMSCs that shared of fibroblastic and histiocytic characteristics in hematopoietic microenvironments of human and mice bone marrow.

Systematic alteration of apoptosis: a review with ultrastructural observations on leukemia cells in vivo.

The ultrastructural characteristics of apoptosis have been described microscopically for four decades. Alterations of nuclei, apoptotic bodies, cytoplasm, and some organelles have been illustrated and investigated during apoptosis. The successive changes of cellular components corresponding with differentiation of apoptotic cells are illustrated in the present review, based on ultrastructural observation of leukemia cells of patients in our routine clinic work by transmission electron microscopy. Most electron micrographs demonstrated that membranous components of nuclear envelop, rough endoplasmic reticulum and Golgi apparatus, and mitochondria were degenerated step by step during apoptosis. The successive images suggested that the endoplasmic reticulum and Golgi apparatus were transferred to cell surface from cytoplasm and participated in formation of apoptotic bodies in apoptosis, although relevant clinical data and more experimental evidence were needed for restraining of leukemia cases from diagnostic work randomly in recent decades.

A novel anemia associated with membranous cytoplasm degeneration in 16 patients: an ultrastructural study.

Sixteen patients with mild anemia and hemolysis were difficult to be classified into any known category based on laboratory examinations and light microscopy. To make a definite diagnosis and investigate the pathomechanism, ultrastructural study was performed on erythroid cells from 16 patients. Transmission electron microscopy demonstrated a series of alterations of cytoplasm, including cytoplasm sequestration, membranous transformation, and degeneration in erythroblasts and reticulocytes at different stages. The affected erythroblasts were usually complicated with chromatin condensation, karyorrhexis, nuclear membrane lysis, and megaloblastic changes. The reticulocytes with the cytoplasm alterations had a huge size from 10 um to 15 um in diameter. The membranous cytoplasm degeneration revealed a unique pathomechanism of dyserythropoiesis and ineffective erythropoiesis in 16 patients with anemia, and suggested a novel anemia category though more details remained to be investigated.

Platelet production of megakaryocyte: A review with original observations on human in vivo cells and bone marrow.

Megakaryocytes (MKs) build characteristic structures to produce platelets in a series of steps. Although mechanisms of demarcation membrane system (DMS) and open canalicular system transformation have been proposed based on experimental studies in recent decades, the related evidence is lacking in human cells in vivo. The present review describes and discusses the development of MKs, transformation of DMS, and the release and maturation of proplatelets based on our observation of human MKs in vivo and bone marrow biopsy by light microscope and transmission electron microscope. Four stages were subdivided from megakaryoblasts to matured cells; presumption of DMS transformation from endoplasmic reticulum and Golgi apparatus were evidenced in contrast to another presumption of DMS transformation from plasma membrane in this review. Effectors of interaction between hematopoietic cells, the sucking and shearing force of sinus blood flow on movement of MKs, and release of proplatelets were emphasized. Additionally, the mechanism of secondary splitting of proplatelets in circulation was demonstrated ultrastructurally. These findings and conceptions might significantly promote our understanding of the mechanism of platelet production in human in vivo cells.

Histiocytic differentiation in acute monocytic leukemia.

Myeloid histocytes of dendritic cells (DCs), Langerhans cells (LCs), and macrophages in varied tissues, as leukemic blasts in acute monoblastic and monocytic leukemia (AML-M5a and M5b), are derived from monocyte progenitors in bone marrow. Based on DC induction from hematopoietic stem cells, myeloid progenitors, and monocytes, and occasional expressions of histocyte-related antigens (HRAs) in M5, we presume some M5 cases share histiocytic phenotypes originally. To clarify the conception, 93 M5 cases were tested with antibodies for HRAs, CD1a, CD163, S100, fascin, and langerin by immunostaining, and their morphologic characteristics were studied by light and transmission electron microscopy. The study revealed that 23 M5 cases were positive for two or more kinds of HRAs and shared a serial of histocytic immunophenotype and morphologic features, which were closely associated with M5b subtype and expression of CD14 in M5.

On the maturation of megakaryocytes: a review with original observations on human in vivo cells emphasizing morphology and ultrastructure.

Megakaryocytes engage in the synthesis of a variety of molecular and macromolecular constituents to build-up characteristic megakaryocyte structure and form proplatelets in a series of cells from megakaryocyte precursors to the fully matured cell. The process is illustrated in this review by light microscope morphology and transmission electron microscopy, which emphasizes new findings in human in vivo megakaryocytes, thereby making a contrast with the abundant literature on megakaryocytes from experimental animal and human in vitro material. Four stages are identified and described, based on the development of characteristic structures including α-granules, dense granules (dense-core granules), the demarcation membrane system (DMS), and proplatelets. The mechanism of DMS development is discussed, in terms of hypotheses suggesting origin from the plasma membrane, and contributions of membrane from the Golgi apparatus and endoplasmic reticulum. The formation of the marginal zone is also discussed, which is suggested to result from a circumscription of the peripheral organelle-free cytoplasmic fringe by peripheral circular cytoskeletal elements such as cytoplasmic actin and microtubules.

Analysis of monocyte and histiocytic cell populations in bone marrow of patients with confirmed and suspected cases of reactive histocytosis.

OBJECTIVE: To describe characteristics of monocytes and histiocytes in the bone marrow of patients with a confirmed and suspected diagnosis of reactive histiocytosis. METHODS: 14 patients with a confident diagnosis of reactive histiocytosis or with a suspected diagnosis were inpatients at the Tianjin Blood Diseases Hospital between 2008 and 2012. Nucleated cells from bone marrow were observed by light microscopy - morphologically and immunohistochemically for histiocyte antigens - and ultrastructurally by transmission electron microscopy. RESULTS: Monocytes, atypical histiocytes, macrophages, hemophagocytes, reticular cells and dendritic cells were significantly increased in 9, 9, 5, 3, 3 and 2, respectively, of the 14 cases. Atypical histiocytes expressed some morphological characteristics of promonocytes. CONCLUSION: Monocytes, atypical histiocytes, macrophages, hemophagocytes, reticular cells and dendritic cells were increased in different relative degrees in patients with bone marrow reactive histiocytosis or suspected reactive histiocytosis. The increase in numbers of monocytes, atypical histiocytes and macrophages was a particularly significant feature. It is argued that atypical histiocytes with immature monocyte features might be precursors of hemophagocytes, reticular cells or dendritic cells.

Ultrastructural characteristics of erythroid cells in congenital dyserythropoietic anemia type II, with a focus on peripheral cisternae and double membranes.

Peripheral cisternae and double membranes (PCDMs) in erythroid cells are a landmark of type II congenital dyserythropoietic anemia (CDA). To gain further insights into the mechanism of dyserythropoiesis, erythroblasts and erythrocytes in bone marrow were studied in 22 Chinese patients with CDA Ⅱ by transmission electron microscopy. The study demonstrated an increase in all patients in erythroblasts with PCDMs with development from pro-erythroblast to red blood cells. PCDMs often connected with cisternae of endoplasmic reticulum (ER) and the perinuclear space, and were accompanied by karyopyknosis, karyolysis and disruption in polychromatic and orthochromatic erythroblasts. The results suggest that PCDMs are transformed from ER during erythropoiesis and participate in the dissolution and deletion of late erythroid cells in patients with CDA II.

Development of Auer bodies from giant inclusions associated with rough endoplasmic reticulum in acute promyelocytic leukemia.

Giant inclusions and Auer bodies in promyeloblasts were investigated in a study which included transmission electron microscopy (TEM) for morphology and ultrastructural cytochemistry for myeloperoxidase in 10 patients with acute promyelocytic leukemia (APL). Ultrastructural cytochemistry demonstrated positive myeloperoxidase reactivity in giant inclusions, expanded rER cisternae, Auer bodies and primary granules. TEM revealed that giant inclusions were adorned by degenerated rER membrane, some of them sharing features with Auer bodies. We hypothesize a novel origin for Auer body development in promyeloblasts of APL, namely that they originate from peroxidase-positive and expanded rER cisternae, and that primary granules were directly released from these expanded rER elements, bypassing the Golgi apparatus.

Ultrastructural alterations of megakaryocytes in thrombocytopenia: A review of 43 cases.

Thrombocytopenia is a frequent occurrence in a variety of hematopoietic diseases; however, the details of the mechanism leading to low platelet count remain elusive. Megakaryocytes are a series of progenitor cells responsible for the production of platelets. Alterations in megakaryocytes in the bone marrow are a causative factor resulting in thrombocytopenia in varied diseases. Based on ultrastructural analysis of incidentally encountered megakaryocytes in 43 patients with blood diseases marked by low platelet counts, electron micrographs demonstrated that aberrant megakaryocytes predominated in idiopathic thrombocytopenic purpura, aplastic anemia, and myelodysplastic syndrome; autophagy, apoptosis, and cellular damage in megakaryocytes were a prominent feature in aplastic anemia. On the other hand, poorly differentiated megakaryocytes predominated in acute megakaryoblastic leukemia (AMKL) although damaged megakaryocytes were seen in non-AMKL acute leukemia. This paper documents the ultrastructural alterations of megakaryocytes associated with thrombocytopenia and reveals distinctive features for particular blood diseases. A comment is made on future avenues of research emphasizing membrane fusion proteins.

Bone marrow mesenchymal cells: polymorphism associated with transformation of rough endoplasmic reticulum.

To understand the behavior and function of bone-marrow mesenchymal cells (BMMCs), we overviewed the morphological presentation of BMMCs in bone-marrow granules (b-BMMCs), isolated BMMCs (i-BMMCs), and BMMCs (c-BMMCs) cultured in H4434 methylcellulose semisolid and MEM media. All samples were derived from bone-marrow aspirates of 30 patients with hematocytopenia. Light microscopy exhibited b-BMMCs and i-BMMCs characterized by abundant cytoplasm and irregular shape in bone-marrow smears, as well as c-BMMCs in culture conditions. Scanning electron microscopy demonstrated cultured c-BMMCs with a sheet-like feature enveloping hematopoietic cells. Transmission electron microscopy revealed b-BMMCs constructing a honeycomb-like structure by thin bifurcate processes among hematopoietic cells. Furthermore, i-BMMCs had bifurcate parapodiums on the surface and prominent rough endoplasmic reticulum (rER) connected with the plasmalemma of the parapodiums. The detailed images suggested that rER may serve as a membrane resource for plasmalemmal expansion in BMMCs in bone marrow.

[Ultrastructure and Raman Spectral Characteristics of Two Kinds of Acute Myeloid Leukemia Cells].

OBJECTIVE: To investigate the Raman spectral characteristics of leukemia cells from 4 patients with acute promyelocytic leukemia (M3) and 3 patients with acute monoblastic leukemia (M5), establish a novel Raman label-free method to distinguish 2 kinds of acute myeloid leukemia cells so as to provide basis for clinical research. METHODS: Leukemia cells were collected from bone marrow of above-mentioned patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 30-50 cells from each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA) and cluster analysis, and the spectra of leukemia cells from 7 patients were analyzed and classified. Characteristics of Raman spectra were analyzed combining with ultrastructure of leukemia cells. RESULTS: There were significant differences between Raman spectra of 2 kinds of leukemia cells. Compared with acute monoblastic leukemia cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks in 622, 643, 757, 852, 1003, 1033, 1117, 1157, 1173, 1208, 1340, 1551, 1581 cm-1. The diagnostic models established by PCA-DFA and cluster analysis could successfully classify these Raman spectra of different samples with a high accuracy of 100% (233/233). The model was evaluated by "Leave-one-out" cross-validation and reached a high accuracy of 97% (226/233). CONCLUSION: The level of macromolecules of M3 cells is higher than that of M5. The diagnostic models established by PCA-DFA can classify these Raman spectra of different cells with a high accuracy. Raman spectra shows consistent result with ultrastructure by TEM.

[The effect of transmission electron microscopy on diagnosis of acute myeloid leukemia].

OBJECTIVE: To analyze coincidence rate of acute myeloid leukemia (AML) sub-typing between transmission electron microscopy (TEM) and clinical discharge diagnosis. METHODS: Reviewing sub-typing results of TEM, light microscopy, flow cytometric analyzing, molecular biological detection and karyotype in 793 AML cases, comparing their coincidence rates with discharge diagnosis to reveal advantages of AML sub-typing by TEM. RESULTS: General coincidence rates of TEM, light microscopy, flow cytometric analyzing, molecular biological detection and karyotype on AML sub-typing were 63%, 59%, 52%, 47%, 26% and 23% respectively, and clinical coincidence rates of TEM on M1, M2a, M4 and M5, M6, M7, t (8; 21) and t (15; 17) were 39%, 34%, 17%, 74%, 50%, 73%, 87% and 89% respectively. CONCLUSION: TEM has a higher coincidence rate in general AML sub-typing, especially strong screenings on t (15; 17), t (8; 21), M7, M5 and M6, but lower coincidence rates on M1, M2a and M4 sub-typing than other methods.

[Correlation between ultrastructural abnormality of bone marrow cells and anemia and neutropenia in myelodysplastic syndrome].

The aim of study was to investigate the relationship of anemia and neutropenia with ultrastructural abnormalities of erythroblasts and young neutrophils in bone marrow of patients with myelodysplastic syndrome (MDS). Anemia parameters and peripheral neutrophil amount of 74 patients with MDS were measured by automatic hemocyte analyzer. According to Hb value and neutropenia degree, MDS patients were divided into 4 groups: normal, mild, middle and severe anemia or neutropenia. The morbid rate and apoptosis rate of erythroblasts and young neutrophils in bone marrow were measured by transmission electron microscopy (TEM). The results indicated that 68 out of 74 patients were consistent with anemia diagnostic criteria, and 51 out of 68 patients were with neutrocytopenia. TEM showed different abnormal features of erythroblasts and young neutrophils in all patients. The morbid rates of erythroblasts in normal, mild, middle and severe anemia groups were 37 ± 14.7%, 24 ± 9%, 32 ± 16% and 34 ± 21% respectively, while apoptotic rates of erythroblasts in normal, mild, middle and severe anemia groups were 2.25 ± 1.03%, 4.43 ± 2.60%, 8.78 ± 4.04% and 11.67 ± 4.57% respectively. The morbid rate and apoptotic rate of erythroblasts were correlated negatively with Hb and HCT value (p < 0.05). The apoptotic rates of bone marrow young neutrophils in 4 groups with different degree of neutropenia were 6.00 ± 2.67%, 9.50 ± 4.42%, 13.00 ± 3.54% and 17.00 ± 2.39%, which correlated negatively with peripheral neutrophil quantity (p < 0.01). Morbid rates of neutrophils in normal, mild, middle and severe anemia groups were 12.25 ± 16.31%, 13.5 ± 10.01%, 23 ± 8.59% and 51.67 ± 19.67% respectively, which positively correlated with its apoptotic rates (p < 0.01). It is concluded that anemia and neutropenia in patient with MDS are correlated with apoptosis and morbid rate of erythroblasts and young neutrophils in bone marrow, which may result in ineffective hematopoiesis.

[Characteristics and clinical significance of CD73 expression in subtypes of leukemia].

The study was purposed to investigate the expression of CD73 on bone marrow nucleated cells (BMMNC) in various leukemia subtypes and its relationship with cell differentiation of leukemia. Immunocytochemistry staining and Wright-Giemsa staining of BMMNC from 75 cases of leukemia, 11 cases of myelodysplastic syndrome (MDS), 13 cases of non-leukemic patients and 9 healthy adults were performed, and the CD73(+) ratio in BMMNC and its relationship with differentiation of leukemia cells were analyzed. The results showed that the ratios of CD73(+) in BMMNC of com-B ALL, pre-B ALL and PLL were significantly higher than those in B-CLL (p < 0.05). CD73(+) ratios in AML subtypes of M(1), M(2a), t (8; 21), t (15; 17), M(4) and M(5) were markedly higher than those in MDS respectively, but in M(6) and MDS were lower and had no statistical difference between them. CD73(+) ratios in T-ALL, B-CLL, M(6), MDS, non-leukemia patients and healthy adults were close to each other and all of them were lower than those in B-ALL and other AML subtypes. It is concluded that the expression of CD73 is associated with leukemia subtype, differentiation and development. The higher differentiation of leukemia cells, the lower of CD73 expression in myeloid and B lymphoid leukemia, but T-ALL does not meet this pattern.