Blood morphology of the European eel (Anguilla anguilla). II. Studies on granulopoiesis]

It was found that the granulocytes for Anguilla anguilla mainly form in the kidney. The stem cell of granulocytes is the large hemocytoblast. Sequences of development were established for the heterophil, cosinophil and basophil granulocytes. Myeloblasts, promyelocytes, metamyelocytes, mature red-shaped and segmented forms were described as stages of development of the heterophil granulocytes. In the case of the eosinophil and basophil granulocytes the myeloblastic and myelocytic stages could be demonstrated as well as the mature granulocyte. By the aid of special granular staining, phase contrast observations, supravital and cytochemical investigations the granulocytes could be described. The cytochemical proof for the granulocytes delivered a distribution pattern of the lipids, carbohydrates, RNA (RNS) and enzymes: unspecific esterase, alkaline and acid phosphatases, oxydase and peroxydase. By means of heart puncture a larger loss of blood was caused and the hematopoiesis stimulated. The following blood letting gave indications of how rapidly the granulopoiesis develops and how much time maturing takes. After a larger loss of blood the new-growth of the granulocytes will be completed nine days later.     

Electron microscopical and histochemical studies of differentiation and function of the cephalopod gill (Sepia officinalis L.)

Summary Phase contrast and electron microscopical investigations on the gill ofSepia officinalis L., from the stage shortly before hatching to the adult stage, demonstrate (1) peripheral respiratory epithelial areas and (2) highly folded epithelia rich in mitochondria in the concave recesses of the gill lamellae. Enzyme-histochemical and cytochemical findings, and in particular the GOT proof, suggest by analogy with the chloride cells of the teleosts that the cells of this second type of epithelium are capable of active transport, and that they probably secrete ammonia. Apparently the gill of cephalopods, too, serves both respiratory and excretory functions.This study was supported by the Deutsche Forschungsgemeinschaft     

团头鲂外周血细胞的显微结构及细胞化学特征

采用常规瑞氏染色和细胞化学染色方法对团头鲂(Megalobrama amblycephala)外周血细胞的显微结构及细胞化学特征进行了观察。在团头鲂外周血细胞中可区分出六类细胞: 红细胞、淋巴细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞和血栓细胞。其中淋巴细胞是除红细胞外含量最多的细胞, 其次分别为血栓细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞。成熟红细胞多为卵圆形, 表面光滑, 胞核呈椭圆形或圆形, 染色质较为致密; 淋巴细胞多呈圆形, 胞质较少, 胞核常偏位; 单核细胞多为圆形, 胞核呈圆形或椭圆形, 胞质内可见空泡状结构; 嗜中性粒细胞近似圆形, 胞核常偏于细胞一侧, 呈分叶状、肾形或椭圆形, 核质界限清晰; 嗜酸性粒细胞一般为圆形, 胞核为肾形或椭圆形, 胞质中充满紫红色颗粒; 血栓细胞形态多样, 主要有椭圆形、纺锤形、长杆状和泪滴形, 核质比较大。淋巴细胞呈α-醋酸萘酚酯酶(ANAE)阳性, 呈过碘酸-雪夫(PAS)、氯乙酸AS-D萘酚酯酶(AS-DCE)弱阳性, 呈苏丹黑B(SBB)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)及过氧化物酶(POX)阴性; 单核细胞呈POX、ACP强阳性, PAS、SBB、AS-DCE和ANAE为阳性, 呈AKP阴性; 嗜中性粒细胞除PAS和ANAE为弱阳性外, 其他染色结果和单核细胞相同; 嗜酸性粒细胞呈POX、ANAE强阳性, SBB、ACP阳性, PAS及AS-DCE则为弱阳性, 呈AKP阴性; 血栓细胞呈PAS、AS-DCE及ANAE弱阳性, 呈SBB、ACP、AKP及POX阴性。团头鲂外周血细胞的显微结构及细胞化学特征与其他鱼类具有相似之处, 但亦有其明显的物种特异性。该研究结果可作为监测团头鲂健康状态的依据, 为其养殖及病理诊断提供基础资料。     

Haematology of swordtail, Xiphophorus helleri. II: cellular composition and isolation of blood cells from kidney of Xiphophorus helleri

The quantitative determination of blood cells in the kidney of swordtail, Xiphophorus helleri , was determined by light microscopical observations. Lymphocytes/thrombocytes and neutro-philic granulocytes (32.4 and 31.6%, respectively) represent the main blood cells in kidney. High amounts of blastic cells in different mitotic phases indicate the role of the kidney as a major haematopoietic organ in this fish species. Additionally, a good and reproducible method for the isolation of accessory and immunoreactive cells was developed using density gradient centrifugation.     

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.     

Nature of human spleen red pulp cells with special reference to sinus lining cells

Summary A histological and cytological as well as enzyme histo- and cytochemical analysis (alpha-naphthyl acetate esterases, naphthol AS acetate esterases, naphthol AS-D chloroacetate esterases, acid and alkaline phosphatases) of human spleen cells in sections and imprints was carried out with special reference to sinus lining cells. These cells show strong naphthol AS esterase activity and no or only little alpha-naphthyl acetate esterase and acid phosphatase activity. Thus they can be distinguished from reticular cells in pulp cords and from other macrophages in cords and sinuses. From the morphological and enzyme histochemical aspect it can be deduced that the sinus lining cells are a distinct cell type of the human spleen. The comparison of these enzyme cytochemical findings with the results of biochemical and electron microscopical investigations suggests that reticular cells of pulp cords and littoral cells of sinuses also have different functions: reticular cells seem to have a high phagocytotic activity while littoral cells seem to be only facultatively phagocytic.This work was supported by the Deutsche Forschungsgemeinschaft.     

长薄鳅外周血细胞的显微结构和细胞化学特征研究

长薄鳅外周血细胞可分为红细胞、中性粒细胞、单核细胞、淋巴细胞和血栓细胞.在数量上,中性粒细胞、单核细胞、淋巴细胞和血栓细胞占白细胞总数的百分比分别是17.06%、5.83%、28.16%和48.94%.细胞化学染色显示所有白细胞均含有糖原物质,所有红细胞均不含酸性磷酸酶,中性粒细胞、单核细胞、淋巴细胞和血栓细胞均含有酸性磷酸酶.非特异件酯酶染色显示单核细胞呈阳性反应,中性粒细胞、淋巴细胞和血栓细胞均为部分呈阳性反应.所有细胞的碱性磷酸酶、过氧化物酶、苏丹黑显色反应均呈阴性.     

Chicken thrombocytes in culture: lymphocyte-conditioned medium delays apoptosis.

Chicken thrombocytes are nucleated cells, analogs to mammalian platelets. These cells are involved in hemostasis, phagocytosis and secretion of specific products. Most of the properties of avian thrombocytes have been established in experiments that employed recently isolated blood cells. Attempts to cultivate these cells for a long period of time under optimal culture conditions for peripheral blood cells were unsuccessful; thrombocytes died after 24 h of cultivation unlike macrophages cocultured with them. Here we investigate the reasons and type of thrombocyte death in culture. Thrombocytes were separated from peripheral blood of roosters and cultured for 48 h. The influence of different culture conditions on thrombocyte viability was studied. Cells were cultured as adherent cell monolayers or under agitation (preventing adherence), in the presence or lack of lymphocytes or their soluble factors, and various concentrations of fetal bovine serum. After 24 h in standard culture thrombocytes displayed cytoplasm and chromatin condensation, DNA cleaved into oligonucleosomal fragments and unaltered mitochondria. These results strongly suggest that thrombocytes suffer an apoptotic cell death in culture. Apoptosis could be delayed by culturing thrombocytes in the presence of lymphocytes or their soluble factors.     

Megakaryoblastic micromegakaryocytic crisis in chronic myeloid leukemia

Atypical megakaryoblasts (MKB) or megakaryocytes (MK) are occasionally present in the peripheral blood during the terminal development of chronic myeloid leukemia (CML). We report on a 49-year-old female suffering from Ph1 chromosome-positive CML with typical megakaryoblastic transformation in the peripheral blood and in the bone marrow. The small "blasts" were at the most only slightly larger and were occasionally even smaller than lymphocytes but showed megakaryoblastic or atypical megakaryocytic differentiation. The cytoplasmic cytochemical pattern of the atypical megakaryocytic cells was identical to that of large atypical thrombocytes. Platelet peroxidase was detected upon electron-microscopic (EM) examination. Immunologic characterization disclosed the presence of MK-specific antigens. When cultured in vitro on agar, the blasts transformed spontaneously into large mature MK, exhibiting characteristic cytochemical and immunological patterns. Cytogenetic examination of peripheral blood showed severe abnormalities. The patient did not respond to therapy and died 3 months after manifestation of the blast crisis.     

The Effect of Contrast Medium SonoVue<Superscript>®</Superscript> on the Electric Charge Density of Blood Cells

The effect of contrast medium SonoVue® on the electric charge density of blood cells (erythrocytes and thrombocytes) was measured using a microelectrophoretic method. We examined the effect of adsorbed H⁺ and OH^? ions on the surface charge of erythrocytes or thrombocytes. Surface charge density values were determined from electrophoretic mobility measurements of blood cells performed at various pH levels. The interaction between solution ions and the erythrocyte’s or thrombocyte’s surface was described by a four-component equilibrium model. The agreement between the experimental and theoretical charge variation curves of the erythrocytes and thrombocytes was good at pH 2–9. The deviation observed at a higher pH may be caused by disregarding interactions between the functional groups of blood cells.     

Haematology of the turbot, Psetta maxima (L.): ultrastructural, cytochemical and morphological properties of peripheral blood leucocytes

Optimum procedures for fish handling and sample processing for use when employing haematological parameters as health indicators in turbot, Psetta maxima (L.), have been established. We found thrombocytes to be the most abundant blood cell, representing approximately 52% of circulating leucocytes (lymphocytes, 40.8%; granulocytes, 5.6%; monocytes, 1.6%; total number of leucocytes=1.3 × 10⁵ ml⁻¹; packed cell volume=22.7%). The light- and electron-microscopical characteristics of these cell types are described, together with their cytochemical properties using Sudan Black B, Periodic Acid Schiff, Non-specific Esterase, and Acid and Alkaline Phosphatase. Turbot thrombocytes showed a high degree of shape alterations when observed in live preparations using phase contrast microscopy, while ultrastructural observations following the in vitro uptake of carbon particles supported an active process of phagocytosis by the thrombocytes, rather than passive entrapment. The lymphocytes of turbot are structurally similar to mammalian lymphocytes with the highest nuclear:cytoplasmic ratio of all the leucocytes observed. Small lymphocytes predominated, large lymphocytes forming less than 1% of the total white blood cell population. The most frequent granulocyte type was a neutrophil-like cell with an eccentric nucleus, only rarely seen in segmented form. In vitro uptake of carbon particles by granulocytes was not observed under the conditions of the experiment, although turbot granulocytes are capable of phagocytosis under different circumstances. These are discussed, along with other physiological and technical factors which can influence the blood parameter findings in fish.     

The cytochemistry of rabbit peripheral blood leukocytes.

The authors report on their investigations of the differentiation between neutrophilic and eosinophilic granulocytes of the peripheral blood in healthy rabbits. By varying the pH-value and the incubation time it is possible to achieve a reliable differentiation between neutrophilic and eosinophilic granulocytes of the rabbit by means of 6 different cytochemical methods which could only be made incompletely with the routine methods used up till now (Eosin-, Giemsa-, Wright's-colouring etc.). Moreover, monocytes and lymphocytes can also be identified reliably.     

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34⁺ hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2–3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.     

Cytochemical analysis of the content of chicken thrombocytes vacuoles.

The content of the large vacuoles present in chicken thrombocytes was analyzed by the use of cytochemical techniques which indicated the presence of basic proteins, unsaturated fatty acids, sugars rich in viccinal hydroxyl groups, linked to proteins or lipids and acid phosphatase. These substances perform one of the most conspicuous functions of these cells, which is phagocytosis. In addition, thrombocytes are committed to hemostasis. Both functions make these cells similar to human platelets, even though having different origins and morphologic characteristics. The large vacuoles described are connected to the open canalicular system (OCS) and together with other cellular structures they contribute to the endocytic system.     

The ultrastructure of the blood cells of the pike Esox lucius L.

The ultrastructure of pike peripheral blood cells, lymphocytes, thrombocytes, granulocytes, and monocytes is described. At present there are no reliable criteria for differentiating between round thrombocytes and small lymphocytes of fish on a routine basis. At the ultrastructural level thrombocytes could be clearly differentiated from lymphocytes by cytoplasmic canals and vesicles, marginal microtubules, and large glycogen deposits. Electron microscopic identification of thrombocytes was confirmed by examining the ultrastructural features of a purified thrombocyte fraction. In addition, a preliminary investigation of the structure of the haemopoietic cells in the thymus, anterior kidney, and spleen was carried out.     

Autoantibodies to the thrombocytes and erythrocytes in HIV-infected patients

In 48 patients with HIV infection were tested for the presence of autologous and allogenic antibodies to red blood cells with the use of Coombs' direct and indirect tests. 18 HIV-infected patients had IgG antibodies to thrombocytes, circulating in the blood (detected by the method of EIA) and bound to thrombocytes (detected by the method of RIA). In 5 out of 48 patients Coombs' direct test yielded positive results with red blood cells. 6 out of 18 examined patients had an elevated content of thrombocyte-bound antibodies. The presence of cross reactions between gp120 of human immunodeficiency virus and gp3a thrombocytes led to the formation of antithrombocyte antibodies and, consequently, to a decrease in the number of thrombocytes.     

Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing

Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed for rapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern.     

A new method for the determination of thrombocyte function in patients and in preserved blood]

In a circulating thrombocytic plasma the pressure in front of a defined mesh filter depends on the combined capacity of adhesion and aggregation of thrombocytes. A new clinical in vitro test is based on this principle which comes nearest to the conditions to be found in hemostasis dependent on thrombocytes. Judging from the preliminary results of investigations performed on stored blood, blood donors, patients with thrombocytopenia, disturbances of blood flow and patients under anticoagulant therapy, the time until the pressure rise after administering ADP and the maximal pressure amplitude proved to be the most significant parameters of the new procedure.     

Haematology of swordtail, Xiphophorus helleri. I: blood parameters and light microscopy of blood cells

The main blood parameters of the swordtail, Xiphophorus helleri , were studied. Morphology, granulation staining and cytochemistry of leucocytes in peripheral blood, kidney, spleen and gills were investigated by light microscopy. Blood parameters are similar to other fish species: Red blood cell count (4.5 × 10⁶μl), white blood cell count (15.2 × 10³μl), haema-tocrit (33.8%) haemoglobin (7.8 mg ml⁻¹), MCV (mean corpuscle volume, 75.1 μm³). MCH (mean content of haemoglobin, 17.3 pg), MCHC (mean percentage haemoglobin/erythrocyte, 23.1%/100 ml erythrocytes). Leucocytes can be classified into lymphocytes, thrombocytes, neutrophilic and eosinophilic gra-nulocytes, monocytes macrophages and melanomacrophages.
Morphological and cytochemical features of the cells are described and compared with results from other fish species.     

Hematologic complications in acute alcoholism]

The authors investigated the relations between cell anomalies (vacuolation, increasing sideroblastosis) caused by the uptake of alcohol and the dynamics of haematopoesis in the bone-marrow of 33 alcoholists who had been admitted in a comatose condition and who were neither affected with anaemia nor with chronic hepatitis. In all cases a maturation arrest of erythropoetic and granulopoetic cell elements which was not in accordance with the number of immature vacuolated cells could be observed. 12 test persons showed increased sideroblastic indices and a slightly diminished medullary reticulocytosis. The majority showed a very active thrombocytopoesis contrasting with the normal or even diminished number of thrombocytes in the peripheral blood. The authors come to the conclusion that alcohol will cause a general metabolic damage of haematopoesis and at the same time it will produce a direct toxic effect on the bone-marrow cells (proerythroblasts, promyelocytes) and the peripheral blood (thrombocytes).