Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) I. Granulocytes

The cytochemistry and staining of granula in peripheral blood granulocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl-acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. Different granula types were shown by triazid-staining (eosinophil and neutrophil granula) and methylenblue-staining for basophil granulation. Toluidinblue-staining was used for basophil granulocytes. Lipids were shown by the Sudan-black-reaction. Four granulocyte subpopulations are described: neutrophil, heterophil, basophil and eosinophil granulocytes. Neutrophils possess all tested granula types, whereas heterophil and basophil granulocytes show only basophil granula. Neutrophils and heterophils show no activity of the tested esterases with the exception of AS-D. Only neutrophils were peroxidase-positive. Alkaline phosphatase and β-glucuronidase were not detected in granulocytes. Basophils and especially eosinophils were rarely found in peripheral blood.     

Ultrastructural and cytochemical observations on the granulocytes of the sturgeon, Acipenser brevirostrum (Chondrostei)

Granulocytes from cranial granulopoietic tissue were studied under the electron microscope, and cytochemistry carried out oncranial and peripheral blood granulocytes of two sturgeons, Acipenser brevirostrum . Ultrastructurally, eosinophils and basophils had homogeneous electron-dense granules similar to those of teleosts and some higher vertebrates. Neutrophils contained two granule types: small elongated fibrillar granules and large (<3.8μm long) usually homogeneous granules.
Neutrophil fibrillar granules were positive for alkaline phosphatase (ALP), acid phosphatase (ACP), α-naphthyl acetate esterase (ANAE), acetyl-l-tyrosine-α-naphthyl esterase (ATNE) and periodic acid Schiff (PAS) reaction. The large homogeneous granules were negative for all enzymes, and were only PAS positive. Eosinophils had granular, cyanide-, azide- and aminotriazole-resistant peroxidase (PO) and were ACP, ATNE, tosyl-l-lysine-α-naphthyl esterase (TLNE) and Luxol fast blue positive.
Ultrastructure and cytochemistry are discussed in relation to other vertebrates, and eosinophils identified as the main phagocytic leucocyte.     

The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

Cytochemical studies of normal feline blood and bone marrow cells

Blood and bone marrow cells of ten clinically healthy cats were stained for alkaline phosphatase (ALP), peroxidase (PO), chloroacetate esterase (CAE), alpha-naphthyl butyrate esterase (NBE), sudanophilia, and periodic acid-Schiff (PAS) reaction. Mature neutrophils in blood and bone marrow were devoid of ALP and NBE, but exhibited modest to strong PO, CAE, sudanophilia, and PAS reaction. In bone marrow, sudanophilia, PO, and CAE were prominent at the promyelocyte stage and diminished with cellular differentiation and maturation, while PAS reactivity increased with cell maturation usually from the myelocyte stage onwards. Myeloblasts were negative for all cytochemical reactions, but some large unidentifiable cells reacted strongly for ALP. Eosinophils were slightly reactive for ALP, CAE, and PAS, but not for PO, sudanophilia, and NBE. Basophil granules stained strongly for CAE, revealed PAS positivity, and stained negatively for PO, NBE, ALP, and sudanophilia. Slight ALP activity was detected in the intergranular cytoplasm of basophils. Lymphocytes and monocytes, with few exceptions, stained negatively. An occasional lymphocyte revealed slight globular NBE activity (NaF-resistant) and diffuse PAS reaction, while an occasional monocyte contained a few PO-positive and sudanophilic granules. Monocytes reacted modestly, whereas bone marrow macrophages reacted strongly for NBE (NaF-sensitive). Cells of the erythroid series stained negatively for all cytochemical reactions, megakaryocytes were PAS-positive, and platelets gave positive reactions for PAS and CAE.     

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。     

A method for the ultrastructural demonstration of non-specific esterase in human blood and lymphoid tissue

Summary Using the substrate 2-naphthylthiol acetate (NTA), we developed a reproducible method of demonstrating a non-specific esterase while retaining nuclear and cytoplasmic details at the ultrastructural level. The NTA esterase had a distribution and pattern of staining similar to those of esterases demonstrable at the light microscopic level by the -naphthyl acetate or naphthol AS-D acetate esterase reaction.The NTA esterase appeared as intensely electron-dense granules of varying size and shape in the cytoplasm. The granules were most abundant in the cells of the histiomonocytic series. The large number of diffusely scattered granules in the cytoplasm of the histiocytes and monocytes made it possible to separate these cells from other haematopoietic elements. There was usually no direct relationship between the NTA esterase positivity and the amount or the location of lysosomes or mitochondria, although in some histoocytes the granules appeared to the associated with lysosomes. The NTA esterase-positive granules were usually more numerous than lysosomes and were located outside the lysosomal granules. Some of the lymphocytes outside the germinal centres and most of the lymphocytes in the blood showed a punctate positivity in the form of 1–4 electron-dense dots. Plasma cells were usually negative but, in rare cases, contained an occasional single dot-like reaction product similar to that in some of the lymphocytes. Granulocytes were always negative.The method described in this paper can be used effectively for identification and study of human haematopoietic cell lines at the ultrastructural level.     

Hematoxylin as a Pituitary Stain

The accurate picture of the acidophil granules of the anterior pituitary which is provided by iron hematoxylin can be combined with the differential staining of the basophils by either the periodic acid-Schiff (PAS) or combined aldehyde-fuchsin-PAS procedures. To accomplish this the two stages of the iron hematoxylin technique are separated so that mordanting in iron alum precedes and application of hematoxylin follows the basophil procedures.     

Sulfated glycosaminoglycans in guinea pig basophils studied by means of cationic colloidal gold

 Bone marrow embedding in the hydrophilic resin, Lowicryl K4M, followed by cationic colloidal gold (CCG, pH 1.0) staining was used to study the sulfated glycosaminoglycans (GAGs) and their sites of sulfation ultrastructurally in various maturational stages of both basophil granulocytes and basophil granules in the guinea pig. CCG at pH 1.0 is specific for sulfated GAG staining. Basophil granulocytes and granules reacted positively to CCG with a variety of staining according to the stage of maturation. The formation of basophil granules takes place throughout the myelocyte stage. Early basophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late myelocytes contain a small and less active Golgi apparatus as judged by CCG staining. All the immature granules and some of the granules with characteristic ultrastructure stained positively. However, some of the mature granules had lost their affinity for CCG upon maturation. Interestingly, strongly positive CCG staining was also observed in the trans to transmost Golgi apparatus. This indicates that sulfation of GAGs occurs in the trans to transmost Golgi apparatus in all maturational stages of basophil granulocytes. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG staining. Accepted: 17 July 1997     

Morphological,cytochemical, and biochemical observations on the blood of the tuatara,Sphenodon punctatus

Abstract

Morphological features of the circulating blood cells of an adult female tuatara are described and illustrated. Mitosis in circulating erythrocytes and the occurrence of an intraerythrocytic haemogregarine parasite are noted. The packed cell volume (haematocrit) was 35%. There were about 310 000 erythrocytes and 7200 leucocytes per mm³ of blood. The lymphocyte was the predominant leucocyte, followed by the eosinophil, monocyte, neutrophil, and basophil. The eosinophils and neutrophils reacted positively to acid phosphatase and peroxidase; the neutrophils were also esterase positive, and all three granulocytes were PAS positive. Haemoglobin, plasma protein, glucose, sodium, potassium, cholesterol, and triglyceride levels were determined. The primitive nature of the tuatara is reflected in the morphological. mensural, and biochemical data presented.     

Light and electron microscopic studies on the enzyme cytochemistry of leucocytes of eels, Anguilla species

The leucocytes of three anguillid eels were studied using enzyme cytochemistry. Leucocytes were stained for peroxidase, alkaline phosphatase, acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, lysozyme, a variety of non-specific esterases, chloroacetate esterase and two proteases. All cells were negative for aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and β-galactosidase. Very few neutrophils, thought to be mature, and all eosinophils contained peroxidase-positive granules, and some monocytes showed very weak peroxidase staining. All leucocytes lacked alkaline phosphatase, but all cells except lymphocytes and thrombocytes of A. dieffenbachii contained acid phosphatase. Neutrophil acid phosphatase released into phagosomes was associated with Escherischia coli bacteriolysis. Neutrophils also secrete lysozyme and, with monocytes, produce and secrete a variety of esterases. The possible interaction of lysozyme, acid phosphatase and esterases in bacteriolysis is discussed.     

Cytochemical study of acute promyelocytic leukaemia.

Five cases of acute promyelocytic leukaemia (A.P.L.) are investigated for peroxidase, PAS, toluidine blue, astra blue, alpha-naphthyl esterase, double esterase incubation (naphthol-AS plus naphthol-AS-D chloroesterase), and cellular lysozyme activity. These cytochemical investigations may contribute further characterization of the morphologic type.     

Ultrastructural cytochemistry of glycoconjugates in basophils from humans and animals

Summary The distribution of glycoconjugates in the basophil granules of humans, guinea pigs, and rabbits was compared. The observation of acid mucopolysaccharides using the dialyzed iron method and of sulfated glycoconjugates using the high iron diamine method revealed three types of reactions in the basophil granules of all three species: (1) granules showing a strong overall reaction, (2) granules showing reaction only at their periphery, and (3) granules showing no reaction. With regard to the relationship between maturation and the types of basophil granules, it appeared that, in general, there were many type-1 granules among immature basophils, but that these granules decreased in mature basophils as type-3 granules increased. The reaction patterns of periodate-reactive neutral glycoconjugates, as shown by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, were different from those of acid mucopolysaccharides: the reaction of basophil granules was diffusely positive, and localization at the periphery was rarely observed. Therefore, unlike the acid mucopolysaccharides, it was difficult to classify the glycoconjugates into three types. However, as with acid mucopolysaccharides, there was a tendency for periodate-reactive glycoconjugates to decrease as maturation progressed. In terms of different species of animals, the reaction of periodate-reactive glycoconjugates with PA-TCH-SP was stronger in humans and rabbits than in guinea pigs.     

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half‐smooth tongue sole Cynoglossus semilaevis

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non‐specific esterase (NSE), peroxidase (POD) and mucous‐cell types was evaluated in the gastrointestinal tract of the half‐smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB‐PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB‐) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS‐AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.     

RNA interference as a gene silencing therapy for mutant MYOC protein in primary open angle glaucoma

Bronchoalveolar lavage (BAL) is a useful diagnostic tool in interstitial lunge diseases (ILD). However, differential cell counts are often non specific and immunocytochemistry is time consuming. Staining of glyoproteins by periodic acid Schiff (PAS) reaction may help in discriminating different forms of ILD. In addition, PAS staining is easy to perform. BAL cells from patients with idiopathic pulmonary fibrosis (IPF) (n = 8), sarcoidosis (n = 9), and extrinsic allergic alveolitis (EAA) (n = 2) were investigated. Cytospins from BAL cells were made and cells were stained using Hemacolor quick stain and PAS staining. Lymphocytic alveolitis was found in sarcoidosis and EAA whereas in IPF both lymphocytes and neutrophils were increased. PAS positive cells were significantly decreased in EAA compared to IPF and sarcoidosis (25.5% ± 0.7% vs 59.8% ± 25.1% and 64.0% ± 19.7%, respectively) (P < 0.05). No significant correlation between PAS positive cells and inflammatory cells was observed. These results suggest that PAS staining of BAL cells may provide additional information in the differential diagnosis of ILD. Further studies ware warranted to evaluate PAS staining in larger numbers of BAL from patients with ILD.     

Ultrastructure of the secretory cells of the submucosal glands in the human maxillary sinus

Tissue samples obtained from the lateral wall of the maxillary sinuses of five patients were examined by light microscopical, histochemical, and ultrastructural techniques. Submucosal glands were tubulo-alveolar mixed glands. The acini consisted of either all serous or all mucous cells, or a mixture of both. Serous granules were stained by toluidine blue, or by hematoxylin and eosin (H and E), but showed little or no reaction with periodic acid-Schiff (PAS) or Alcian blue. Mucous granules were pale in toluidine blue or H and E preparations, and consisted primarily of acid mucosubstances, as demonstrated by their staining reaction with PAS and Alcian blue. At the electron microscope level, the serous granules were either homogeneously dense, or showed a substructure consisting of at least two layers of distinctly different electron-opacity. Typical mucous droplets consisted of a fibrillar network dispersed in a translucent matrix. A second secretory product was present in the mucous cells in the form of elongated, membrane-bounded structures containing numerous parallel filaments, which measured about 55 Å in diameter. The mucous droplets and the filamentous bodies appear to arise from the opposite faces of the Golgi complex in the mucous cells. The filamentous bodies showed a pronounced tendency to fuse with the mucous droplets. All acini were surrounded by a well-defined myoepithelial layer and contained intercellular nerve terminals.     

American paddlefish leukocytes demonstrate mammalian‐like cytochemical staining characteristics in lymphoid tissues

American paddlefish Polyodon spathula leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, α‐naphthyl butyrate esterase and β‐glucuronidase. American paddlefish monocytes, lymphocytes and granulocytes stained positive for acid phosphatase. Monocytes stained positive for α‐naphthyl butyrate esterase. Lymphocytes that stained positive for α‐naphthyl butyrate esterase were designated type A. Lymphocytes that stained positive with antibodies to the L chain of white sturgeon Acipenser transmontanus immunoglobulin (Ig) were designated type B. Type A and type B lymphocytes stained positive for β‐glucuronidase. All leukocytes observed were negative for Sudan Black B. Monocytes, lymphocytes and granulocytes were present in the renal haematopoietic tissue, spleen, thymus, pericardial myeloid tissue, lamina propria of the spiral valve, and in meningeal myeloid tissue located dorsal to the brain, at the base of the brain and around the notochord. Peyer's patches were present in the gut. Morphological characteristics of leukocytes stained with Wright's and haematoxylin and eosin and appeared very similar to those of other fish species.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Characteristics on staining of leukocytes in experimental animals]

The staining characteristics of the peripheral blood cells from mouse, rat, guinea pig, rabbit, dog, marmoset and monkey were studied. In marmoset, it is easy to distinguish neutrophils from eosinophils by using the phosphate-buffered solution of pH 5 or 6. It was found in the special staining methods that neutrophil granules showed intense peroxidase and Sudan black B reactions in marmoset in comparison with those in the other species of experimental animals. Neutrophil granules rabbit was, however, intensely stained with esterase and acid phosphatase.     

Commercial Hair Sprays as Fixatives for Hematological Cytochemistry

Commercial hair sprays have been found to be excellent cytological fixatives for a variety of enzymatic and nonenzymatic hematological staining procedures. Of the varieties evaluated, not all were found suitable for each staining procedure tested. With some preparations, excellent leukocyte morphology and preservation of reaction product was obtained after staining for carbohydrates (PAS), lipid (Sudan black), nucleic acids (methyl green-pyronin), peroxidase, M-nadi oxidase and alkaline phosphatase. These spray preparations are remarkably inexpensive, readily stored, and stable and simple to use. The fixative ability is probably related to their polyvinylpyrrolidine and alcohol content.