Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake (Elaphe obsoleta quadrivitatta)

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake are described. A panel of cytochemical stains, including demonstration of myeloperoxidase, acid phosphatase, naphthol AS-D chloracetate esterase, alpha-naphthol butyrate esterase and alkaline phosphatase activities; and periodic acid-Schiff and Sudan Black-B staining was performed. Snake heterophils lacked peroxidase, alkaline phosphatase and acid phosphatase activity. Azurophils stained positively for all stains except alkaline phosphatase activity. Lymphocytes showed positive acid phosphatase activity. Differentiation of thrombocytes from lymphocytes was very difficult even with cytochemical staining. Only a minor staining difference was observed with periodic acid-Schiff stain. Thrombocytes exhibited coarse, dark, purple stippling usually located in the polar area of the cytoplasm, whereas lymphocyte staining varied from none to very fine, pale pink granules dispersed throughout the cytoplasm. Ultrastructural characteristics were similar to those of mammalian leucocytes with the exception that the snake basophil granules have no crystalline matrix, and heterophil granules appeared as large, elongate, membrane bound structures of varying density with no distinct core or matrix.     

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake (Elaphe obsoleta quadrivitatta)

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake are described. A panel of cytochemical stains, including demonstration of myeloperoxidase, acid phosphatase, naphthol AS-D chloracetate esterase, alpha-naphthol butyrate esterase and alkaline phosphatase activities; and periodic acid-Schiff and Sudan Black-B staining was performed. Snake heterophils lacked peroxidase, alkaline phosphatase and acid phosphatase activity. Azurophils stained positively for all stains except alkaline phosphatase activity. Lymphocytes showed positive acid phosphatase activity. Differentiation of thrombocytes from lymphocytes was very difficult even with cytochemical staining. Only a minor staining difference was observed with periodic acid-Schiff stain. Thrombocytes exhibited coarse, dark, purple stippling usually located in the polar area of the cytoplasm, whereas lymphocyte staining varied from none to very fine, pale pink granules dispersed throughout the cytoplasm. Ultrastructural characteristics were similar to those of mammalian leucocytes with the exception that the snake basophil granules have no crystalline matrix, and heterophil granules appeared as large, elongate, membrane bound structures of varying density with no distinct core or matrix.     

In vivo leucocyte interactions on Pellethane surfaces

In vivo leucocyte interactions of three Pellethane materials of varying hardness were qualitatively and quantitatively characterized using a cage implant system over a 21 d implantation period. Scanning electron microscopy (SEM) and cytochemical staining were utilized to observe the cellular events occurring at the leucocyte-biomaterial interface. Many of the quantitative assays performed, the intracellular alkaline phosphatase activity of exudate leucocytes, the intracellular acid phosphatase activity of adherent leucocytes, the density of adherent leucocytes and the foreign body giant cell network formation tendencies of adherent leucocytes, suggest increased cellular activation with increased Pellethane hardness. Qualitative SEM evaluation of Pellethane surfaces revealed a variety of cellular activities. These included macrophage adherence, cytoplasmic spreading and macrophage-macrophage membrane fusions to form foreign body giant cells. The foreign body giant cells exhibited nuclear reorganization and, when compared with adherent macrophages, they displayed an enhanced ability to fuse to neighbouring leucocytes, increased spreading of membrane processes over the polymer surface, the presence of large cytoplasmic vacuoles, and a lengthened duration of enzymatic activity. Contact angle analysis showed the Pellethane surfaces to be hydrophobic and of low hysteresis. The critical surface tension and the dispersive component of the total surface tension were found to increase with Pellethane hardness.     

Alkaline and Acid Phosphatase in Murine Leukemia

Alterations for acid and alkaline phosphatase levels and their pattern of splenic and lymph node activity in normal and virus-induced lymphoblastic leukemia were studied. Enzyme levels were examined by using both cytochemical and biochemical procedures. The GC leukemia virus, a ribonucleic acid murine virus antigenically related to the Rauscher-Moloney viruses, was used to stimulate acid and alkaline phosphatase by producing lymphomaceous disease in Ha/ICR mice. With the Burstone and Gomori cytochemical procedures, both enzymes were found in higher than normal levels in lymphomaceous spleen and lymph nodes. Confirmation of the cytochemical studies was obtained by enzyme assay of cell-free homogenates in each case with the exception of spleen acid phosphatase. The discrepancy between the cytochemical tests which showed significant elevation of spleen acid phosphatase and the enzyme assays which failed to reveal such elevation could be due to a labile acid phosphatase isozyme which is lost on cellular disruption during homogenate preparation. A significant spleen alkaline phosphatase specific activity elevation above normal was found with a 50% incidence only when leukemic spleen wet weight increased nearly threefold its normal value. This result suggests that alkaline phosphatase elevation is a secondary event occuring after the onset of disease and is not a fundamental metabolic alteration concerned with the onset of murine lymphoblastic leukemia.     

Cytochemical profile of megakaryoblastic leukaemia: a study with cytochemical methods, monoclonal antibodies, and ultrastructural cytochemistry.

A cytochemical study using: Sudan black B; alpha-naphthyl acetate (ANAE) staining; estimation of alpha-naphthyl butyrate (ANBE) esterase activity; acid phosphatase activity; and 5' nucleotidase activity was carried out in 15 cases of megakaryoblastic leukaemia. These included cases of M7 acute myeloid leukaemia and blast crises of chronic granulocytic leukaemia. The megakaryoblastic nature of the blasts was first established using two monoclonal antibodies against platelet glycoproteins, and by estimating the platelet/peroxidase reaction at ultrastructural level. Our findings suggest that megakaryoblasts have a typical cytochemical profile comprising positive ANAE staining and acid phosphatase activity with a predominant localisation in the Golgi zone and negative or weak ANBE activity. A similar positive cytochemical pattern was also found in five cases of erythroleukaemia (M6). The specificity of the 5'nucleotidase activity for megakaryoblasts was not confirmed. In most cases of megakaryoblastic leukaemia there was no 5'nucleotidase activity only two cases showed positive reactions--reactions were positive in several cases of myeloblastic and lymphoblastic leukaemia. We suggest that cytochemical methods may be useful in diagnosing M6 and M7 acute leukaemia because less than 40% of leukaemic cells react with specific monoclonal antibodies.     

Ultrastructural morphology and cytochemistry of iron-deficient polymorphonuclear leukocytes

Previous studies have documented decreased activities of certain enzymes and altered function in polymorphonuclear leukocytes (PMN) during iron deficiency. The present study was undertaken to determine if the enzymatic abnormalities could be correlated with morphologic or quantitative change in PMN granules. Ultrastructural examination of primary and secondary granules and assessment of the secondary granule components alkaline phosphatase and vicinal glycol-containing glycoconjugates was performed in rabbit bone marrow, peripheral blood, and peritoneal heterophils. In addition, biochemical quantifications of the secondary granule component alkaline phosphatase and the primary granule marker beta-glucuronidase were performed. The results confirmed that a marked, significant decrease in alkaline phosphatase occurs in iron-deficient animals; however, no biochemical decrease in beta-glucuronidase activity was observed. Ultrastructurally, PMN secondary granules of iron-deficient rabbits tended to be more numerous than in controls when examined with morphometric and glycoconjugate staining methods, but lacked staining in alkaline phosphatase preparations. These results demonstrate that iron-deficient rabbits produce normal to increased quantities of primary and secondary granules, despite a uniform deficiency of alkaline phosphatase, a secondary granule marker.     

Apical mitochondria-rich cells in the human epididymis: An ultrastructural,enzymohistochemical, and immunohistochemical study

An ultrastructural, enzymohistochemical, and immunohistochemical study of the ductus epididymis in normal men was undertaken to investigate the characteristics of the apical mitochondria-rich cells (AMRCs). These cells, which differ morphologically from the principal cells (PCs), appear in isolation in the caput epididymidis (5.8 ± 1.7 cells per cross-sectional duct) and only occasionally in the corpus epididymidis. The morphologic appearance of AMRCs varies from slender cells extending from the basement membrane to the lumen to apical cells without apparent contact with the basement membrane. The former display a round pale nucleus located in the middle of the epithelium; the apical cells have a dark nucleus, which, surrounded by a narrow cytoplasmic band, protrudes into the lumen. The cytoplasm of AMRCs is electron-dense and contains numerous mitochondria surrounded by rough endoplasmic reticulum cisternae. In the apical portion, there are lysosomes, vesicles with an electron-dense granule, and vacuoles showing a variable size and content. The stereocilia are shorter and less numerous than those of the PCs. The AMRCs are similar to the PCs in the intensely positive reaction for the enzymatic activity acid phosphatase, as well as in the lack of reaction for alkaline phosphatase and phosphorylase activities. AMRCs differ from PCs in: (1) a more intense reaction to the enzymatic activities ATPase, NADP, and succinic dehydrogenease, (2) a more intense immunostaining by AE1/AE3 and Ks4.62 anti-cytokeratin antibodies, and anti-estradiol receptor protein (D5) antibodies, and (3) a lower staining affinity for epithelial membrane antigen (EMA) antibodies. No positive immunostaining for the anti-cytokeratin Ks8.6 antibodies was observed in either AMRCs or PCs.     

Lysosomes in Aortic Smooth Muscle Cells: Effects of Hypertension

Hypertension induces hypertrophy and increased turnover of aortic smooth muscle cells along with an accumulation of connective tissue in the aortic wall. We identified the lysosomes in normal and hypertensive aortic muscle cells by light and electron microscopy, utilizing cytochemical staining for acid phosphatase activity. Lysosomes were found to be more numerous in hypertensive vessels. Biochemical assays of two specific lysosomal enzymes revealed a doubling of acid phosphatase and a more than threefold increase in β-N-acetyl-glucosaminidase activities in hypertensive aortas.     

Apical mitochondria-rich cells in the human epididymis: an ultrastructural, enzymohistochemical, and immunohistochemical study.

An ultrastructural, enzymohistochemical, and immunohistochemical study of the ductus epididymis in normal men was undertaken to investigate the characteristics of the apical mitochondria-rich cells (AMRCs). These cells, which differ morphologically from the principal cells (PCs), appear in isolation in the caput epididymidis (5.8 +/- 1.7 cells per cross-sectional duct) and only occasionally in the corpus epididymidis. The morphologic appearance of AMRCs varies from slender cells extending from the basement membrane to the lumen to apical cells without apparent contact with the basement membrane. The former display a round pale nucleus located in the middle of the epithelium; the apical cells have a dark nucleus, which, surrounded by a narrow cytoplasmic band, protrudes into the lumen. The cytoplasm of AMRCs is electron-dense and contains numerous mitochondria surrounded by rough endoplasmic reticulum cisternae. In the apical portion, there are lysosomes, vesicles with an electron-dense granule, and vacuoles showing a variable size and content. The stereocilia are shorter and less numerous than those of the PCs. The AMRCs are similar to the PCs in the intensely positive reaction for the enzymatic activity acid phosphatase, as well as in the lack of reaction for alkaline phosphatase and phosphorylase activities. AMRCs differ from PCs in: (1) a more intense reaction to the enzymatic activities ATPase, NADP, and succinic dehydrogenease, (2) a more intense immunostaining by AE1/AE3 and Ks4.62 anti-cytokeratin antibodies, and anti-estradiol receptor protein (D5) antibodies, and (3) a lower staining affinity for epithelial membrane antigen (EMA) antibodies. No positive immunostaining for the anti-cytokeratin Ks8.6 antibodies was observed in either AMRCs or PCs.     

Practical immunocytochemical identification of human blood cells

A practical immunocytochemical method of demonstrating surface antigens of human blood cells on air-dried smears or other cytologic preparations has been developed. This method uses monoclonal antibodies as the primary antibodies and calf intestinal alkaline phosphatase as the enzymatic indicator. Combined staining with cytochemical stains for myeloperoxidase or nonspecific esterase on the same slide is also possible when needed. These methods are very useful for accurate identification of human blood cells on the commonly available clinical specimens and are very helpful in the diagnosis and classification of various hematologic neoplasms, including chronic lymphocytic leukemias, acute leukemias, and related diseases.     

Distribution patterns of orthogonal arrays and alkaline phosphatase in plasma membranes of satellite cells in rat spinal ganglia

Summary The plasmalemmal structure of satellite cells in the lumbar spinal ganglia of rat was examined by freeze-fracture and by a cytochemical method for the demonstration of alkaline phosphatase activity. Plasma membranes of satellite cells are the only ones in the ganglia to contain, in addition to globular intramembrane particles, orthogonal arrays of particles 6–7 nm in diameter. The arrays are most concentrated in the portions of the membranes contacting the basal lamina, or outer membranes; they decrease considerably in number in lateral membranes, and are rare in the membrane regions adjacent to the neuronal perikaryon, or inner membranes. Such gradual decrease in array density in satellite cells suggests regional differences of plasma membrane properties within the same cell. Alkaline phosphatase, which was chosen as a cytochemical marker for membrane activity because of its relation to transport function, localizes to inner and lateral membranes, and not to outer membranes of satellite cells. The absence of correlation between localization of orthogonal arrays and such enzymatic activity suggests that the membranes provided with many arrays possess some characteristics different from other membranes that may exclude transport activity. The possible significance of orthogonal arrays and their close association with the basal lamina are discussed.     

Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies

Simultaneous detection of multiple tissue epitopes with an overlapping distribution pattern by monoclonal antibodies is sometimes needed for routine immunohistological evaluations. Therefore, multistep double and triple immunoenzymatic methods using antibodies from the same species or Ig (sub)class have been developed. Since only commercially available monoclonal antibodies (either unlabelled, biotinylated or as fluorescein conjugate) have been used, the techniques may be regarded as generally applicable. The staining protocol for double staining consists of six incubation steps: (1) unlabelled monoclonal antibody 1; (2) enzyme I-conjugated anti-mouse Ig; (3) normal mouse serum--for blocking; (4) fluoresceinated monoclonal antibody 2; (5) rabbit anti-fluorescein isothiocyanate--employing the fluorochrome as hapten; (6) enzyme II-conjugated anti-rabbit Ig. For enzymes I and II, peroxidase, alkaline phosphatase and beta-galactosidase can be applied; excellent results were obtained with the following colour combinations: peroxidase activity in red/alkaline phosphatase in blue and beta-galactosidase in green/alkaline phosphatase in violet. Moreover, this double staining method can be extended to provide an immunoenzyme triple staining technique by mixing biotinylated monoclonal antibody 3 and avidin-biotin enzyme III complex with the steps 4 and 5 reagents, respectively. In this way three tissue epitopes can simultaneously be detected clearly and selectively in green (beta-galactosidase), blue (alkaline phosphatase) and red (peroxidase).     

Correlations between enzymatic and immunologic properties of human peripheral blood mononuclear cells. I. Ectoenzymes of normal and immunodeficient peripheral blood mononuclear cells

The activity of plasma membrane marker enzymes which are involved in purine metabolism (5'-nucleotidase, alkaline 5'-nucleotide phosphodiesterase), in active ion transport (Na-K-Mg-adenosine triphosphatase, ouabain-sensitive Na-K-adenosine triphosphatase), in aminoacid transport (gamma-glutamyltranspeptidase), and in basic physiologic functions (alkaline phosphomonoesterase) were assayed in mononuclear cells isolated from peripheral blood of normal donors and of patients with primary immunodeficiency. Irrespective of the clinical classification of the immunodeficiency, the cells of patients were characterized by significantly diminished 5'-nucleotidase and to a certain extent by lower alkaline phosphomonoesterase activities. Average activity levels of other enzymes were similar in cells of patients and controls, but scattering was more pronounced in the first group. Determination of substrate affinity revealed different kinetic properties of 5'-nucleotidase in cells from patients and normal donors; however, the extent of inhibition by beta-glycerophosphate or alpha, beta-adenosine-methylene diphosphate was comparable for both types of cells. The presence of inhibitory compounds in patients' serum was excluded by mixing experiments. When activities of the various plasma-membrane-associated enzymes were compared with each other, significant correlations emerged in normal lymphocytes. Most of these correlations were absent in cell membranes of immunodeficient patients. The findings indicate that the plasma membrane of lymphocytes from patients with immunodeficiency may be characterized by an altered distribution of enzymatic constituents.     

Cellular location of streptolysin O.

Streptolysin O was measured in subcellular fractions of group A streptococci obtained after preparation of protoplasts in a hypertonic buffer containing raffinose. Most of the activity was located in the periplasm (the region between cell wall and membrane) and did not differ in several characteristics from that of extracellular streptolysin O. Of the enzymes used as subcellular markers, aldolase and maltase (cytoplasmic) and acid phosphatase (membrane associated) were in the same fractions as found in other bacteria. However, the location of alkaline phosphatase differed from that of other bacteria in the most of the activity was in cytoplasm rather than in the periplasm.     

Diagnostic significance of levamisole-resistant alkaline phosphatase in cytochemistry and immunocytochemistry

The authors have used two immunoalkaline phosphatase methods to study nonhematopoietic tumor tissues of four patients, one each with alveolar cell carcinoma of the lung, renal cell carcinoma, gastric adenocarcinoma, and colon carcinoma. They found, regardless of specific antibodies used, definite enzyme activity in the tumor cells of these four patients. Although it was possible to determine that the tumor cells were epithelial in origin because of their intense staining with antibodies to epithelial cell antigens, control slides labeled with nonimmune mouse ascites also contained cells with definite enzyme activity. In two of these cases, unlabeled smears were stained for alkaline phosphatase and showed that the tumor cells contained endogenous levamisole-resistant enzyme activity. This endogenous enzyme activity is not demonstrable in either the benign cells of these cases or the benign or malignant cells of other control cases. The findings suggest that the immunoalkaline phosphatase methods also have their inherent endogenous enzymic problems. They also suggest that cytochemical demonstration of levamisole-resistant alkaline phosphatase may be a useful cell marker for the identification of tumor cells in serous effusions.     

A cytological study of intestinal absorption in the suckling rat

The distal small intestine of the albino rat has the capacity to absorb protein and particulate matter during the suckling period. Ultrastructural and cytochemical aspects of this absorptive phenomenon were examined in the ileum. Soon after the initial ingestion of milk, a large, yellow, smooth membrane-limited, protein body appears in the immediate supranuclear region of ileal absorptive cells and, also, many small vacuoles and membrane-limited droplets arise between this body and the microvilli. Exogenous protein enters an elaborate superficial tubular system and is segregated in membrane-limited vacuoles and droplets and, then, appears in the supranuclear body. The body and adjacent membrane-limited droplets are basophilic, periodic acid-Schiff positive, and rich in hydrolytic enzymes (acid phosphatase, ATPase, thiamine pyrophosphatase, alkaline phosphatase, esterase). The results suggest the presence of a highly developed lysosomal system during the period of protein absorption. Additional cytological features of the absorptive cells are presented. Ileal absorptive cells are normally free of lipid droplets. When emulsified lipid is introduced into the neonatal ileum, it enters into the cytoplasmic smooth membrane system, including the supranuclear body, and later appeas in the lacteals. This suggests both that the uptake of material may be non-selective and that similar intracellular pathways may be used in transporting protein and lipid through the epithelium.     

Disorganization of glycolytic and gluconeogenic pathways in skeletal muscle of aged persons studied by histometric and enzymatic methods

Histometric data obtained by the point counting method, and the enzyme patterns of glycolysis, gluconeogenesis, fatty acid degradation and energy transfer have been determined in the same muscle specimens of m. vastus lateralis from 12 untrained patients between the ages of 4 and 78 years who suffered no disturbance of the neuromuscular system. Activities of 18 enzymes have been related to pure muscle weight corrected for fatty and connective tissue content, as well as to single fibre weight. A comparable muscle enzyme pattern was found in persons of around 20 years old and around 70 years old when expressed per gram of single fibre weight. However, in terms of grams of pure muscle weight, a significant activity decrease with age was obtained for 6-phosphofructokinase, triosephosphate dehydrogenase and phosphoenolpyruvate carboxykinase, whereas activity of hexose diphosphatase increased with age as also did 3-hydroxyacyl-CoA dehydrogenase activity. Five other cytoplasmic enzyme activities involved in glycolysis and energy transfer did not change significantly with age, nor did lysosomal acid phosphatase. The mitochondrial enzyme activities of gluconeogenesis (for example, pyruvate carboxylase, malic enzyme) were diminished to a lesser extent as also the auxiliary enzymes glutamic-oxaloacetic transminase and glutamic-pyruvic transaminase; glutamate dehydrogenase activity remained unchanged. The findings indicate a distinct disorganization of cytoplasmic glycolysis and gluconeogenesis pathways in presenile human skeletal muscle, confirming the histometric data already described. They cannot be explained by changes with age in numerical or areal ratio of type I and type II fibres.     

Influence of the Indigenous Microbiota on Amounts of Protein, DNA, and Alkaline Phosphatase Activity Extractable from Epithelial Cells of the Small Intestines of Mice

Germfree mice housed in isolators under controlled environmental and nutritional conditions were associated with an intestinal microflora. These associated animals and germfree mice drawn from the same population were used in experiments in which saline extracts of cells from the small intestine were assayed for alkaline phosphatase activity and for protein and DNA content. Epithelial cells were harvested from the intestines sequentially from the villous tips to the crypts of Lieberkühn. In all preparations, germfree animals yielded from one and one-third to one and one-half times the mass (wet weight) of cells yielded by associated mice. Likewise, for all preparations, extracts of the mass of cells from germfree mice contained more protein and DNA per milliliter than did extracts from associated animals. The ratio of the amount of extractable protein or DNA per milliliter of extract to the total wet weight of the cells in milligrams was about the same, however, for preparations from germfree and associated animals. All preparations from germfree animals yielded higher total alkaline phosphatase activities than those from associated mice. When related to the amount of DNA in the cells, the enzymatic activities were slightly but not significantly higher in preparations from germfree animals, except for preparations of cells removed from the tips of the villi. When related to the amount of protein in the extracts, the enzymatic activity (i.e., specific activity) was about the same in preparations from germfree and from associated mice, except (again) for preparations of cells removed from the tips of the villi. In the latter preparations, the specific alkaline phosphatase activities and enzymatic activities calculated relative to the amount of DNA were substantially higher for germfree animals than for mice with a microflora. Individual intestinal epithelial cells from germfree and associated animals, except those close to the villous tips, contain about the same alkaline phosphatase activity. Therefore, germfree mice must yield more activity of such microvillous enzymes than do mice with a microflora, partly because enterocytes at the tips of the villi in germfree mice contain more protein with enzymatic activity than do cells in a comparable location in mice with a microflora. In addition, the small intestines of germfree mice contain more activity of enzymes such as alkaline phosphatase than do those of associated animals because the small intestines of the former animals contain more enterocytes than do those of the latter.     

A combined cytochemical and immunocytochemical method for simultaneous visualization of cytoplasmic enzyme reactivity and cell surface antigens in cell suspensions

Immunocytochemical methods were used in combination with enzyme cytochemistry to visualize simultaneously cytoplasmic enzyme reactivity (for dipeptidyl[amino]peptidase [DAP IV], acid phosphatase [AcP], chloroacetyl esterase [CAE]) and cell surface antigens (Leu-3a, Leu-4, Leu-14, Leu-M1, OKT4, OKT8, OKB7) in cytospin preparations from cell suspensions of human reactive lymphoid tissues (four lymph nodes and three tonsils). Different fixative solutions were tested. Enzyme and immunocytochemical reactions were carried out in different orders of sequence to establish which was the better direction for the combination of the two methods. The following immunocytochemical methods were tested: three stages, avidin-biotin complex, peroxidase-antiperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) (using both peroxidase and alkaline phosphatase as labeling enzyme). Acetone or buffered formalin acetone gave the best results both for cytochemical and immunologic reactions. DAP IV and AcP reactivities could be visualized only when cytochemical reactions were performed before immunocytochemistry. CAE reactivity could be demonstrated either before or after immunocytochemistry. Cell surface antigens could be demonstrated with most immunocytochemical methods: however, the APAAP method was preferred for its sensitivity and effectiveness when combined with enzyme cytochemistry. By this approach, cells expressing only immunologic markers and cells expressing only cytochemical markers could easily be distinguished from those coexpressing both markers, because cytochemistry and immunocytochemistry could be combined without affecting the reactivity of each marker, and the reaction products did not hamper the interpretation of preparations.