Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta)

A morphologic classification based on the cytochemical characteristics of blood cells of 35 juvenile loggerhead sea turtles (Caretta caretta) is described. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff, and toluidine blue. The morphologic characteristics of erythrocytes were similar to those reported in green turtles. Six types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes, monocytes and thrombocytes. Except for the basophils, the rest of the white blood cells from loggerhead turtles had different cytochemical characteristics compared to blood cells from other sea turtle species. The leukocyte differential count was different from that reported for other sea turtle species. Heterophils were the most numerous leukocytes from these loggerhead turtles, followed by lymphocytes, eosinophils, monocytes and basophils. This paper provides a morphologic classification of blood cells of loggerhead sea turtles that is useful for veterinary surgeons involved in sea turtle conservation.     

Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies

Five monoclonal antibodies (MAb) produced against cell surface antigens on bovine mononuclear phagocytes (MPh) were characterized. None of the MAb recognized erythrocytes, thrombocytes, B lymphocytes or resting or activated T lymphocytes. Two MAb (IL-A22 and IL-A24) reacted with the majority of monocytes and granulocytes in peripheral blood, with 20-40% bone marrow cells comprising myelo-monocytic cells, and with a proportion of mature macrophages. Reactivity of the remaining three MAb was restricted to MPh: one of these (IL-A25) was apparently specific for pulmonary macrophages, whereas the molecules recognized by the other two (IL-A23 and CH16A) were expressed on subpopulations of blood monocytes and tissue macrophages. None of the MAb inhibited adherence of MPh to plasma-coated gelating surfaces or Fc-mediated rosette formation. One of the MAb, IL-A24, which reacts with MPh and granulocytes, inhibited antigen-specific proliferative response or peripheral blood mononuclear leukocytes (PBM) to the soluble antigen, keyhole limpet hemocyanin (KLH) but did not inhibit responses to concanavalin A or allogeneic leukocytes. This MAb was shown to react with two polypeptides of approximately 75 kD and 110 kD on the surface of peripheral blood monocytes.     

Hematology of dairy cows with purulent-necrotic processes in the digital area treated with Subtilin ointment

The objective of this study was to investigate the effect of Subtilin ointment on the hematology of dairy cows when used to treat inflammation and necrotic tissue in the digital area. Holstein-Friesian cows were allocated to 2 groups of 10 animals each. The first group consisted of clinically healthy cows and the second group consisted of cows with a purulent-necrotic lesion in the digital area. To make a comparative analysis, blood samples of healthy and diseased cows were taken and tested before treatment with Subtilin ointment and 10 days after treatment. The blood of the diseased cows had more leukocytes (53.38%), lymphocytes (46.81%), erythrocytes (15.78%), granulocytes (64.56%), and platelets (34.76%), and a higher mean cell volume (of 8.37%) and increased level of monocytes/eosinophils (65.12%) than did the blood of clinically healthy cows. At the same time, there were no evident changes in the level of erythrocytes, hemoglobin, mean cell hemoglobin, mean cell hemoglobin concentration, and percentage of lymphocytes in either clinically healthy or diseased cows. The hematology of diseased cows treated with Subtilin ointment demonstrated positive dynamics in the healing of ulcerative processes.     

Counting absolute number of lymphocytes in quail whole blood by flow cytometry

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.     

南极企鹅外周血细胞初步研究

2012年8—10月,在天津海昌极地海洋世界采用Wright—Giemsa’s染色和Natt—Herrick’s染色方法研究了南极企鹅（Pygoscelisantarcticus）外周血细胞。研究表明,外周血液分类为：红细胞、白细胞和血栓细胞。其中自细胞包括：异嗜性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴细胞和单核细胞。测得红细胞平均大小（长径短径）为15．32μm×7．83μm,异嗜性粒细胞直径为（11．98±0．97）μLm,嗜酸性粒细胞直径为（11．61±0．78）μm,嗜碱性粒细胞直径为（11．87±0．69）μm,淋巴细胞直径为（9．93±1．29）Ixm,单核细胞直径为（15．04±0．93）μm,血栓细胞直径为（6．96±0．79）μm。统计红细胞总数为（1．85±0．17）×1012／L,白细胞总数为（6．37±1．68）×109／L。     

Bovine mononuclear phagocytic cells: identification by monoclonal antibodies and analysis of functional properties

Monoclonal antibodies (mAb) which react with bovine monocytes have been produced. These include three mAb (P8, IL-A22 and IL-A24) that recognize the majority of monocytes and granulocytes in peripheral blood; two of these mAb were also shown to react with 30-40% of cells in bone marrow, including both monocytic and granulocytic cells, and with variable percentages of tissue macrophages. Thus these mAb can act as markers for myeloid cells in haemopoietic tissues and for monocytes in cell populations devoid of granulocytes. A further two mAb (IL-A23 and IL-A25) recognize monocytes and/or macrophages. The reactivity of one of these mAb (IL-A25) appears to be mainly restricted to pulmonary macrophages. The other mAb reacts with a variable proportion of blood monocytes and generally with a higher percentage of tissue macrophages, suggesting that its expression may relate to activation or maturation of monocytes. In order to study the functional properties of peripheral blood monocytes, techniques were developed for obtaining populations of peripheral blood mononuclear cells (PBM) depleted of monocytes to less than 0.2% and monocyte populations of greater than 97% purity. Removal of monocytes from PBM abrogated the capacity of the cells to proliferate in response to Con A and PBS, although addition of 2-mercaptoethanol to the cultures restored proliferation. In both allogeneic and autologous mixed leukocyte cultures (MLC), monocytes were required in the stimulator cell populations for induction of the proliferative responses, and both responses could be elicited with purified monocytes. However, proliferation in the autologous MLC occurred only with responder cell populations that were depleted of monocytes. Moreover, it was shown that addition of more than 5% unirradiated monocytes to the autologous MLC suppressed proliferation. These findings indicate that monocytes play an important role in the induction and regulation of cellular immune responses in cattle. Two of the mAb that react with monocytes and granulocytes were tested for their capacity to inhibit proliferative responses of PBM to mitogens, alloantigens or the soluble antigen, KLH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unraveling avian and reptilian hematology: An optical and electron microscopic study of the buffy coat

Background

Blood centrifugation and buffy coats are at the cornerstone of hematology. In mammals, the buffy coat has a layered disposition (from bottom to top) with neutrophils on top of erythrocytes, followed by monocytes/lymphocytes, and platelets. In nonmammals, this distribution is unknown. Recently, the cell tube block (CTB) technique was developed to study the buffy coat, but it was never applied to nonmammal buffy coats.

Objectives

This study aimed to evaluate using the CTB technique to study reptilian and avian buffy coats and to propose its use for clinical applications.

Methods

Blood from five birds and eight reptiles of different species was obtained to make CTBs that were processed for optical/electron microscopy. H&E, Sirius red, and immunohistochemistry staining against CD3 (to label T lymphocytes) were applied to the CTBs.

Results

In birds, the buffy coat had a layered appearance with the granulocyte layer containing granulocytes (heterophils and eosinophils) and nucleated erythrocytes followed by a mononuclear cell layer containing lymphocytes, monocytes, and thrombocytes. In some animals, a nucleated erythrocyte layer was observed admixed with the granulocyte/mononuclear cell layer. A small clot within the buffy coat was seen in seven reptiles, and less definition of layers occurred in reptiles, with only one or two layers. Lymphocytes appeared toward the top of the buffy coat.

Conclusions

From a comparative hematology perspective, the buffy coat of mammals differs from that of birds and more from that of reptiles. The CTB technique can be used to study these differences in avian and reptilian hematology, especially to study atypical circulating cells, hemoparasites, or blood cell proportions in health and disease.     

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.     

Ultrastructural characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta)

Ultrastructural characteristics of erythrocytes, heterophils, eosinophils, lymphocytes, monocytes and thrombocytes of the loggerhead sea turtle (Caretta caretta) were evaluated, using blood samples from 15 healthy juvenile animals. Except for the eosinophils, the rest of the white blood cells from loggerhead turtles had similar ultrastructural characteristics compared with blood cells from other sea turtle species. Eosinophils from loggerhead turtles were homogeneous in size, and no crystalline structures were observed within the granules. This paper provides an ultrastructural characterization of blood cells of loggerhead sea turtles, as a reference for future haematological studies of this species.     

Histograms of Complete Blood Counts in Dogs: Maximizing Diagnostic Information

Histograms, which are an integral part of the automated complete blood count, are now available through most of the automatic hematology analyzers used in veterinary clinical practice. Data concerning the size and number of blood cells are graphically presented in histograms, and their variations are also illustrated. Important information that is not apparent from numerical results are sometimes provided by histograms. Histograms are also referred to as frequency distribution curves and are essentially graphs resulting from the placement of the sizes of cells on the x-axis and the number of cells on the y-axis. Typically, automated analyzers provide histograms for each class of blood cells, that is, for erythrocytes, leukocytes, and platelets. Thus, when the erythrocyte histogram shows asymmetry with a right shift, it means the size of the erythrocytes is greater than normal (macrocytosis); when it presents a left shift, the size of the erythrocytes is less than normal (microcytosis). When two peaks are found in the curve, two populations of erythrocytes coexist, as in the case of a blood transfusion or therapeutic response. In the leukocyte histogram, three peaks are found: the closest to the y-axis (left) corresponds to the lymphocytes, the middle to the monocytes, and the right to the polymorphonuclear cells (neutrophils, eosinophils, and basophils). Finally, in platelet histogram, asymmetry with a right shift suggests the presence of giant platelets or schistocytes. Although the histogram is not recommended as a stand-alone test, it allows the practitioner to observe abnormalities in the distribution curve that correspond to abnormalities in the size or number of cells, and to quickly make diagnostic or therapeutic decisions that are particularly important in emergencies.     

Identification of feline monocytes and neutrophils as effector cells in antibody-dependent cellular cytotoxicity: sequential analysis, using light microscopy, histochemistry, and scanning electron microscopy

Feline monocytes and neutrophils functioned as effector cells in antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken erythrocytes. Using light microscopy, effector cell populations were identified in effector-target cell interactions, with further characterization of these identical individual effector cells by histochemical evaluations and scanning electron microscopy. Monocytes and neutrophils, but not lymphocytes, were observed attacking target cells. Carbonyl iron depletion of monocytes and neutrophils from peripheral blood leukocytes caused a marked reduction from a mean of 62% to 3.6% lysis in ADCC as measured by a 4-hour 51Cr release assay. Effector cells functioning in the ADCC reaction were visualized, using sequential analysis and light microscopy, histochemistry, and scanning electron microscopy.     

Characterisation of the green turtle’s leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity

Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27–38 %; 39–45 %) and OVA-Alexa (35–46 %; 14–22 %) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species.     

A new method for counting of quail leukocytes by flow cytometry

An automatic counting method was developed for fish blood cells using a fluorescent dye, 3, 3-dihexyloxacarbocyanine (DiOC (6)(3)), that selectively stain lipid bilayers in living cells. In the present study, the DiOC(6)(3) method was applied to quail (Cotumix cotumix japonica) blood cells. After quail blood cells were stained with DiOC(6)(3), absolute counts and relative proportions of erythrocytes, granulocytes, monocytes, and lymphocytes plus thrombocytes in whole blood were obtained by means of flow cytometry (FC). The number of each cell types by the FC was in good agreement with those counted microscopically. This method will offer new possibilities for routine blood cell counting for avian medicine.     

Acute monocytic leukemia in an irradiated Beagle.

A purebred female Beagle dog that had received 2,000 R of protracted wholebody gamma-irradiation from 60Co when 14 months old had hematologic changes consistent with a myeloproliferative disorder 3 years after the termination of radiation exposure. Peripheral blood and bone marrow findings during the 7-month period before death showed progressive anemia with increased numbers of platelets; immature granulocytes, monocytes and promonocytes. A period of partial remission occurred during which time the peripheral blood was aleukemic, although there was marked thrombocytosis and abnormal erythropoiesis which was evidenced by bizarre circulating nucleated red cells, anisocytosis, poikilocytosis and Howell-Jolly bodies. The dog had a terminal crisis with marked leukocytosis, most cells in the peripheral blood being bizarre monocytes and promonocytes. Tissues obtained at necropsy showed diffuse as well as focal infiltration of the spleen, liver, lymph nodes, heart, kidney and gastrointestinal wall with immature neoplastic cells resembling monocytes and monocytic precursors. The monocytic differentiation of the invasive cell population was confirmed by morphological, cytochemical, histological, ultrastructural and in vitro cell culture studies.     

Morphological and Cytochemical Studies of Peripheral Blood Cells of Schizothorax prenanti

The morphological and cytochemical studies of peripheral blood cells of Schizothorax prenanti were studied by light and electron microscopy. Erythrocytes, thrombocytes and three types of leucocytes, lymphocytes, neutrophils and monocytes, were distinguished and characterized. In addition to mature erythrocytes, immature and dividing erythrocytes were observed. A few organelles such as mitochondria were distributed in the cytoplasm of erythrocytes. Lymphocytes with heavily clumped heterochromatic nucleus and minimal cytoplasm were classified into small and large lymphocytes. Three different populations of granules, with distinctive ultrastructural aspect, were observed in neutrophils. Monocytes were the fewest leucocytes possessing rich organelles, phagocytized materials and vacuoles. Thrombocytes with various types were the most abundant blood cells among leucocytes and contained a prominent nucleus with dense bands of heterochromatin and many cytoplasmic vacuoles. Periodic acid‐Schiff staining was positive in neutrophils, monocytes, lymphocytes and thrombocytes, but not in erythrocytes. Peroxidase‐positive staining was observed in neutrophils and monocytes, but not in erythrocytes, lymphocytes and thrombocytes. Only neutrophils were positive for oil red O. Except for erythrocytes, the other blood cells stained positively for acid phosphatase. Only neutrophils and monocytes were positive for α‐naphthyl acetate esterase. None of the cells studied were positive for alkaline phosphatase. The morphologic and cytochemical features of blood cells of S. prenanti are similar to those of other fish. This investigation may be helpful as a tool to monitor the health status of cultured S. prenanti and will grant early detection of clinical pathology.     

In vitro properties of diffuse cytoplasmic esterase-positive canine mononuclear leukocytes

Depletion of cytoplasmic esterase-positive canine peripheral blood monocytes from mononuclear cell suspensions was attempted using plastic adherence, carbonyl iron ingestion and/or Sephadex G-10 filtration. An esterase-positive, nonadherent, nonphagocytic subpopulation was identified and further characterized by the presence or absence of cell membrane receptors for the Fc portions of immunoglobulin and the activated third component of complement. The majority of these nonadherent cells lacked these receptors. The data suggests that canine peripheral blood monocytes are a heterogenous cell population.     

Ultrastructural and Cytochemical Properties of Peripheral Blood Cells of Piebald Naked Carp (Gymnocypris eckloni)

The ultrastructural and cytochemical properties of peripheral blood cells of Gymnocypris eckloni were investigated by transmission electron microscopy and a range of cytochemical techniques to provide clear insight into the structure and function of blood cells from this fish. Ultrastructurally, erythrocytes, leucocytes (neutrophils, eosinophils, lymphocytes, monocytes), thrombocytes and plasma cells were identified in the peripheral blood of G. eckloni. The most special ultrastructural characteristics of blood cells in this fish were that neutrophils exhibited only one type of cytoplasmic granules containing an eccentric, spherical or oval electron‐dense core, and eosinophils presented two types of granules with non‐uniform electronic density and without crystalloids in their cytoplasm. Neutrophils, eosinophils, lymphocytes, monocytes and thrombocytes were positive for periodic acid–Schiff and α‐naphthyl acetate esterase staining. Intense peroxidase positive staining was observed in neutrophils and monocytes, but not in eosinophils, lymphocytes and thrombocytes. Neutrophils, eosinophils and monocytes were stained positively for acid phosphatase, whereas lymphocytes and thrombocytes did not stain. Leucocytes and thrombocytes were negative for alkaline phosphatase and Sudan black B staining. Erythrocytes were negative for all cytochemical staining. The cytochemical and ultrastructural features of peripheral blood cells of G. eckloni were similar to those of other fish species. However, some important differences were identified in G. eckloni.