Cytochemical staining characteristics of chicken heterophils and eosinophils

Cytochemistry was used to differentiate chicken heterophils and eosinophils in blood smears and cytospin preparations of isolated leukocytes. Cytochemical staining also was performed to evaluate any alteration of cell staining characteristics after heterophils had been isolated using discontinuous Ficoll-Hypaque gradients. Cytochemical staining reactions of heterophils and eosinophils were the same before and after discontinuous gradient cell separation. Characteristic positive reactions did not occur when chicken heterophils were stained with alkaline phosphatase, peroxidase, Sudan black B, acid phosphatase, alpha-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase, periodic acid-Schiff, or Luna's eosinophil granule techniques. Chicken eosinophils stained positively when peroxidase, Sudan black B, acid phosphatase, and Luna methods were performed. In highly cellular cytospin preparations, peroxidase reactions readily distinguished the few contaminating eosinophils (< 1%) from the heterophils.     

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。     

Morphologic, cytochemical staining, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes (Crotalus adamanteus)

OBJECTIVE: To evaluate light microscopic, cytochemical, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes. ANIMALS: 10 healthy snakes. PROCEDURE: Various stains, including Wright-Giemsa, benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff with diastase, and toluidine blue, were used to stain leukocytes differentially on multiple blood smears. Electron microscopy also was performed. RESULTS: Lymphocytes were the most commonly observed leukocyte and could be distinguished from thrombocytes, using periodic acid-Schiff stain with diastase. Azurophils also were commonly observed; their granules stained with peroxidase. Eosinophils were not identified; however, 2 morphologic variations of heterophils were seen in the blood of all snakes and were considered the same cell type at different stages of cytoplasmic granule development. Heterophil granules were better preserved, using a one-step Wright-Giemsa method that did not require alcohol fixation prior to staining. Degranulated heterophils were observed in all preparations. CONCLUSIONS: Most leukocytes of eastern diamondback rattlesnakes can be identified easily on Wright-Giemsa-stained preparations. However, hematologic stains that do not require alcohol fixing prior to staining may be preferred for leukocyte evaluation in certain reptiles. A limited degree of heterophil maturation may continue in the blood of healthy snakes. This, along with degranulation of heterophils, may result in a variable staining pattern in this cell type, regardless of the stain used. CLINICAL RELEVANCE: Results provide baseline data for use in hematologic testing in diagnosis of disease and monitoring of treatment of sick or injured snakes.     

Identification and quantification of granulocytes in caecal mucosa and submucosa of chickens experimentally infected with Eimeria tenella and Salmonella enteritidis

1. Poultry granulocytes are not clearly distinguished from each other with haematoxylineosin (HE) stain; thus, histochemical techniques must be used. Three experiments were carried out using 4-week-old Leghorn chickens. 2. Three, 80-chicken groups were orally infected with (1) 10 8 colony forming units (CFUs) Salmonella enteritidis , or (2) 10 4 Eimeria tenella oocysts, or (3) 10 8 CFUs S. enteritidis + 10 4 E. tenella oocysts. Ten chickens from each group were euthanased and caecum samples obtained. Caecum samples were fixed in 10% formalin (buffered, pH 7.4) at 4, 8, 12 h, 1, 3, 5, 7, and 14 d post-inoculation (PI). 3. Samples were stained using three different staining techniques: HE for the identification of heterophils and eosinophils, Ziehl-Neelsen for mast cells, and p -phenilenediamine dihydrochloride plus pyrocatechol (PPD + PC) for eosinophils. 4. Birds from Experiment 1 showed no changes in the numbers of granulocytes. Birds from Experiments 2 and 3 showed higher numbers of heterophils in caecal mucosa and submucosa separately, on d 5 and 7. In Experiment 3, a decrease was observed in submucosal mast cells on d 3. Chickens from Experiments 2 and 3 showed increased numbers of mucosal mast cells between d 7 and 14. 5. PPD + PC positively stained eosinophils, but not heterophils. 6. Numbers of heterophils and mast cells were increased during the acute inflammatory process caused by E. tenella . Therefore, mast cells could play a role as primary inflammatory cells. Eosinophils seem not to be part of the inflammatory process caused by E. tenella .     

Separation of turkey heterophils from blood using two-step Ficoll-Hypaque discontinuous gradients

A method is presented to separate turkey heterophils from anticoagulated whole blood using two-step Ficoll-Hypaque discontinuous gradients and ammonium chloride lysis of contaminating erythrocytes. Heterophils can be isolated from multiple blood samples within 3 to 4 hours. Using this technique, 66.4 +/- 18.4% (mean +/- standard deviation) of blood heterophils were harvested. Final cell isolates averaged 96.0 +/- 2.9% heterophils with few contaminating eosinophils (2.5 +/- 2.3%) or basophils (1.6 +/- 1.8%). Cell viability, as determined by trypan blue dye exclusion, was 98.0 +/- 1.4%.     

Light microscopic study of granulocytes of Pygoscelid penguins (Pygoscelis adeliae) of Antarctica

Morphologic features of heterophils, eosinophils, and basophils of wild adult Pygoscelid penguins (Pygoscelis adeliae; 1 male, 4 females) of Antarctica were studied. Granulocytes were described on the basis of analysis of stained blood films and of 0.5-micron sections of centrifuged resin-embedded blood. Morphometric analysis of granulocytes included average cell diameter, cell shape, and nuclear configuration as well as the number, size, and shape of intracytoplasmic granules. Large round heterophils were the most prevalent granulocyte and possessed numerous rod- and spindle-shaped granules and a polymorphous nucleus. The second most prevalent granulocyte was the eosinophil. These eosinophils contained a bilobed nucleus and an average of 29.52 homogeneous granules/cell. The smallest and least encountered granulocyte was the basophil. Basophils possessed a single eccentric nucleus surrounded by round metachromatic granules (12.20 granules/cell) of varying size.     

A study of manatee leukocytes using peroxidase stain

Leukocytes of the manatee, dog, cat, horse, rabbit, bird, and human were stained with modified Wright-Giemsa stain and myeloperoxidase stain. The predominant segmented leukocyte of the manatee stained positive for myeloperoxidase in a manner similar to the human, dog, cat, and horse neutrophil. Rabbit and bird heterophils stained unlike the manatee predominant leukocyte with myeloperoxidase stain. Eosinophils of all species examined also stained differently from the predominant manatee leukocyte. We conclude that the predominant segmented cell type of the manatee is not biochemically similar to the heterophil of the bird and rabbit or to the eosinophil of the human, dog, cat, horse, bird, and rabbit.     

Morphologic characterization of specific granules in Greyhound eosinophils

BACKGROUND: "Vacuolated" eosinophils (ie, eosinophils with empty, nonstaining granules) have been described previously in normal Greyhounds. However, to our knowledge, detailed studies of granules in vacuolated and normal eosinophils in this breed have not been performed. OBJECTIVE: The objective of this prospective study was to characterize some of the morphologic, ultrastructural, and cytochemical staining features of specific (primary) granules in both normal and vacuolated eosinophils in Greyhound blood. METHODS: Morphologic features of eosinophils in Wright's- and Diff-Quik-stained peripheral blood smears from 49 Greyhounds were compared with 200 blood smears from non-Greyhound dogs. Transmission electron microscopy was done on blood from 3 Greyhounds with vacuolated eosinophils and 3 with normal eosinophil granules. Blood smears from 4 of these dogs also were stained cytochemically with alkaline phosphatase (AP), chloracetate esterase (CAE), and alpha naphthyl butyrate esterase (ANBE). The morphologic features and tinctorial properties of vacuolated and normal eosinophils were compared. RESULTS: Twenty-six Greyhounds (53%) had vacuolated eosinophils and 23 (47%) had normal granulated eosinophils in smears stained with Wright's stain. Only 1% of eosinophils were vacuolated in non-Greyhound dogs. Twenty of the 23 (85%) Greyhounds with normal granulated eosinophils on Wright's-stained smears had vacuolated eosinophils in smears stained with Diff-Quik. Ultrastructurally, no morphologic differences were observed between granules of vacuolated and normal eosinophils. Both vacuolated and normal eosinophils in Greyhounds were positive for AP and negative for CAE and ANBE, as expected for normal dogs. CONCLUSION: Vacuolated eosinophils in Greyhounds likely reflect, at least in part, differential staining properties of the specific granules with different hematologic stains. Ultrastuctural and cytochemical features of eosinophil granules were similar in normal and vacuolated eosinophils from Greyhounds.     

Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 1. Evaluation of cytological stains and the percentage of mast cells and eosinophils

OBJECTIVE: To compare a fast Romanowsky cytological stain (Diff-Quik) and Leishman's stain for the detection of mast cells in samples from the lower airways of racehorses, and to compare the proportion of mast cells and eosinophils in the total inflammatory cells in tracheal aspirate (TA) with those in paired bronchoalveolar lavage (BAL) samples. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses. PROCEDURE: Fifty-one paired TA and BAL samples were collected after treadmill exercise from 48 horses with poor racing performance. Two slides were prepared from each sample; one was stained with Diff-Quik stain and the other with Leishman's stain. Differential cell counts of eosinophils and mast cells were recorded from each slide. Comparison of the suitability of the stains for the detection of mast cells, and comparisons of eosinophil and mast cell percentages in TA and BAL samples were analysed using the non-parametric Wilcoxon matched pairs test. RESULTS: Percentages of mast cells were significantly higher in Leishman than in Diff-Quik stained slides in both TA (P = 0.03) and BAL samples (P < 0.0001). Mast cell percentages were significantly higher in BAL than in TA samples using Leishman's stain (P < 0.0001). There was no significant difference in eosinophil percentages between TA and BAL samples (P = 0.07). CONCLUSIONS: Fast Romanowsky type stains (for example Diff-Quik) are not appropriate for the detection of mast cells in samples from the equine lower respiratory tract. Therefore, a metachromatic stain that reliably identifies mast cells (for example Leishman's) should be used if evaluation of mast cells in lower respiratory tract is undertaken. Mast cells are predominantly found in the distal small airways and alveoli sampled with a BAL. In contrast, eosinophils appear to be evenly distributed in the lower respiratory tract. However, high percentages of eosinophils are occasionally found only in TA samples. We recommend that both a TA and BAL be used for the evaluation of eosinophils and mast cells within the equine lower respiratory tract.     

Morphology,cytochemical staining and ultrastructural characteristics of reindeer (Rangifer tarandus) leukocytes

Peripheral blood smears from four adult reindeer (Rangifer tarandus) were examined after staining with Romanowsky's stain and cytochemical stains, including alpha-napthyl butyrate esterase (alpha-NBE), Sudan black B (SBB), chloroacetate esterase (CAE) and alkaline phosphatase (ALP). Romanowsky-stained eosinophils, neutrophils, lymphocytes and monocytes resembled those of cattle, sheep and goats. Basophils had two different staining patterns with Romanowsky's stain. Basophils that we termed "grey basophils" were similar in appearance to grey eosinophils in Greyhound dogs, with medium blue-grey to lavender-grey cytoplasm containing varying numbers of clear vacuoles or granules and variable numbers of small, intensely basophilic, perinuclear granules. The second basophil staining pattern was more typical of ruminant basophils, with uniform, pale to dark basophilic cytoplasmic granules. Basophils stained positive for alpha-NBE, SBB, CAE, and ALP. Eosinophils stained positive for SBB, and were negative for alpha-NBE, CAE, and ALP. Neutrophils were negative for SBB, CAE, and ALP. Monocytes stained positive for alpha-NBE, were rarely positive for CAE and SBB, and were negative for ALP. Transmission electron microscopy revealed matrix within all granulocytes granules, including those of basophils.     

Separation of avian heterophils from blood using Ficoll-Hypaque discontinuous gradients

Rapid separation of avian heterophils from anticoagulated whole blood was achieved using Ficoll-Hypaque discontinuous gradients. An average of 14.4% of blood heterophils was harvested with a mean purity exceeding 99%. Heterophil viability, as determined by trypan blue dye exclusion, averaged 99.8%. The integrity of isolated heterophils was evaluated by cytochemical staining and ultrastructural examination. Cytochemical staining reactions of heterophils in whole blood and of isolated cell suspensions were similar. No ultrastructural abnormalities were observed. Using this procedure, viable intact heterophils were rapidly isolated from blood with an acceptable cell yield and purity for cell function studies.     

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.     

Estimation of platelet counts on feline blood smears

Blood samples were obtained from 50 cats admitted for hematologic evaluation at the Royal (Dick) School of Veterinary Studies. Manual platelet counts were done using a hemacytometer, and the average number of platelets per oil immersion field (1,000X magnification) was determined on stained blood smears. A hemacytometer count was not obtained for one sample because of a failure in erythrocyte lysing. In nine samples, obvious platelet clumps in the blood smear prevented accurate determination of the number of platelets per oil immersion field. Hemacytometer counts on these nine samples ranged from 260-587 X 10 (3) platelets/microliter, suggesting that platelet clumps on a blood smear were usually associated with adequate platelet numbers. Simple regression analysis of hemacytometer counts and the average umber of platelets per oil immersion field for the remaining 40 samples yielded correlation coefficients (r) of 0.776 on untransformed data, and 0.892 on log10-transformed data. Each platelet per oil immersion field represented a circulating platelet count of approximately 20 X 10(3)/microliter, similar to conversion factors reported for dogs and human beings. It was concluded that estimation of platelet number on stained blood smears is a simple and quick method that appears to be reliable over a wide range of platelet counts in cats.     

Normal haematopoiesis,cellular components and stainable iron content in the bone marrow of camels (Camelus dromedarius)

Bone marrow samples were collected from the ribs of 20 healthy adult male Iranian camels (Camelus dromedarius). The bone marrow smears were stained using Wright's stain. Blood samples were collected at the same time for routine haematological examination. The development and morphology of the blood cells in the bone marrow of the camels were similar to those of other domestic species. The mean myeloid/erythroid ratio was 1.21, the mean erythroid percentage was 42.7% and the mean myeloid percentage was 52.0%. The mean percentage of other cells was 5.3%. The mean percentage of eosinophils in the myeloid series was higher than in other domestic species. Iron stores, estimated from Perl's stain, ranged from scant (1+) to moderate (3+) but most samples had 2+ iron content. All peripheral blood results were within reference ranges.     

Hematology and clinical chemistry of adult yellow-headed temple turtles (Hieremys annandalii) in Thailand

Background: Yellow‐headed temple turtles (YHT), Hieremys annandalii, native to Thailand, are protected from exploitation under the Wild Animal Reservation and Protection Act, also listed under Appendix II of the Convention on International Trade of Endangered Species and the International Union for the Conservation of Nature red list. Objectives: The objectives of this study were to describe quantitative, morphologic, and cytochemical features of blood cells and plasma biochemical analytes of clinically healthy YHT. Methods: Blood samples were collected from 40 adult YHT from October 2007 to February 2008. Hematologic and biochemical analyses, cytochemical staining, and ultrastructural evaluation were performed using standard methods. Results: Hematologic results (mean ± SD) included: RBC count, 0.275 ± .094 × 10⁶ cells/μL; WBC count, 11.7 ± 6.6 × 10³ cells/μL; heterophils, 29.4 ± 6.9%; eosinophils, 23.7 ± 5.3%; basophils, 21.2 ± 1.9%; lymphocytes, 14.8 ± 5.9%; and azurophils, 10.7 ± 5.3%. Erythrocytes stained dark red with peroxidase‐staining. Periodic acid‐Schiff stain could not differentiate between thrombocytes and lymphocytes. Thrombocytes contained cytoplasmic vacuoles, similar to mammalian platelets and those of birds and snakes. Heterophils and eosinophils were similar in structure and cytochemical staining characteristics to those of other turtles and reptiles. Structure of basophils was similar to avian basophils. Lymphocytes and azurophils had similar cytochemical staining compared with mammalian lymphocytes and monocytes. Mean MCHC, WBC counts, absolute azurophil counts, and plasma alanine aminotransferase activity were higher in male turtles than in females. Conclusion: Blood characteristics of YHT are species‐specific, and this study can be served as a reference for future clinical studies and medical care of YHT.     

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.     

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.     

Light microscopic and ultrastructural characterization of cells recovered by respiratory-tract lavage of 2- and 6-week-old chickens

This study characterized the cell population recovered by respiratory-tract lavage of 57 two-week-old and 59 six-week-old specific-pathogen-free chickens as a prerequisite to study the response of the avian respiratory tract to infectious agents. The respiratory tract of each bird was lavaged through the trachea with a series of three lavages of 10 ml of room-temperature, neutral phosphate-buffered saline per lavage. The three lavages per bird were pooled for analysis. Total recovery volumes were measured, lavage fluid cellularity was determined, and a 200-cell differential count of non-erythrocyte cells was performed. Lavage fluid recovery was greater from 2-week-old birds (91.3 percent) than from 6-week-old birds (86.3 percent). Total cells recovered were greater for 6-week-old chickens (6.79 x 10(5)) than for 2-week-old chickens (5.03 x 10(5)). Cells of epithelial origin included squamous cells, goblet cells, and both ciliated and non-ciliated columnar epithelial cells. Cells of non-epithelial origin consisted of heterophils, lymphocytes, macrophages, eosinophils, basophils, and erythrocytes. Cells of epithelial origin were the predominant cell type recovered from the 2-week-old chickens, followed by heterophils. In 6-week-old chickens, heterophils were the predominant cell type recovered, followed by cells of epithelial origin. In descending order of prevalence, the remainder of cell types recovered from chickens of both ages were lymphocytes, macrophages, eosinophils, and basophils.