C. I. acid red 52: a new stain for cells of granulocytic origin

Using the xanthene dye C.I. acid red 52 (C.I. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52. In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

Identification of normal and leukemic granulocytic cells with merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Identification of Normal and Leukemic Granulocytic Cells with Merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytos and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in crythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single a p t stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Preferential Staining of Granulocytic Cells by Sulphonaphthyl Red

Using sulphonaphthyl red (Michrome #947), granules in mature and immature granulocytic cells stained bright red. Granules were not visualized in other types of normal or leukemic blood cells. As such, sulphonaphthyl red may be useful in distinguishing normal and abnormal granulocytic cells from other types of blood and bone marrow cells.     

Preferential staining of granulocytic cells by sulphonaphthyl red

Using sulphonaphythyl red (Michrome #947), granules in mature and immature granulocytic cells stained bright red. Granules were not visualized in other types of normal or leukemic blood cells. As such, sulphonaphthyl red may be useful in distinguishing normal and abnormal granulocytic cells from other types of blood and bone marrow cells.     

ULTRASTRUCTURAL LOCALIZATION OF DIALYZED IRON-REACTIVE MUCOSUBSTANCE IN RABBIT HETEROPHILS, BASOPHILS, AND EOSINOPHILS

For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 µ, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.     

Identification of lymphocyte subpopulations with a polymethine dye

Using the polymethine dye p-ethoxyphenyl-p-aminostyryl-1,3,3-trimethyl-3H-indolium chloride as an aqueous stain applied to specimens of peripheral blood or buffy coat fixed in FAA fixative, differential coloration of leukocytes was achieved using darkfield illumination. Neutrophils stained dark maroon and contained green granules, eosinophils contained bright blue granules, basophils revealed yellow and pink granules, and monocytes stained green with green and yellow vacuoles. In studies of purified lymphocyte subpopulations obtained in a cell sorter, T-helper cells stained red, T-suppressor cells were yellow orange, B-cells appeared yellow and often contained yellow annular structures in the cytoplasm, and natural killer (NK) cells stained green and contained large green granules. As a rapid screening technique for identification of T-helper and T-suppressor cells and their ratios in health and disease, the new polymethine stain may complement the more complex monoclonal antibody techniques for identification of these cells.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Which granules can be stained with azure B and eosin?

The standardized stain composed of pure azure B and eosin, as published by Wittekind and colleagues in 1986, demonstrated granules in neutrophilic leucocytes that were much coarser than those seen after staining with conventional Romanowsky-Giemsa methods. These granules belong to at least two classes. Their identification cannot be achieved by means of the morphologic characteristics of single granules; a multivariate analysis of the granulation as a whole, and a comparison with specifically stained primary granules is required. In particular, this study on unbiased cell samples showed that with Wittekind's method, the primary granules in peripheral neutrophils are stained. Further study of clinical smears revealed an enhanced dye uptake by the secondary granules. The staining behavior of the granules is related to the leukocyte count.     

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.     

A combined stain for identifying epithelial cells of the gastric mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: paradoxical concanavalin A staining (PCS) to identify mucous neck cells, periodic acid Schiff-concanavalin A staining to distinguish mucous neck cells from surface mucous cells, and a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: Feulgen hydrolysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; Feulgen hydrolysis-PAS-concanavalin A-Bowie staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears

Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of 'toxic' granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).     

Cholesterol-free phospholipid domains may be the membrane feature selected by N epsilon-dansyl-L-lysine and merocyanine 540

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.     

A Combined Stain for Identifying Epithelial Cells of the Gastric Mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: A) paradoxical concanavalin A staining (PCS) to identify mucous neck cells, B) periodic acid Schiff-concana-valin A staining to distinguish mucous neck cells from surface mucous cells, and C) a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: 1) Feulgen hydroIysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; 2) Feulgen hydrolysis-PAS-concanavalin A-Bowic staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.     

Merocyanine 540 as a fluorescent probe of membranes: selective staining of leukemic and immature hemopoietic cells.

We have reported (Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate of failure in membrane maturation during leukemic blood cell development.     

Immunocytochemical localization of lactoferrin in human neutrophils

Summary The subcellular localization of lactoferrin in human neutrophils was studied by an electron-microscopic immunoperoxidase method. This molecule was detected in small granules of blood polymorphonuclear leukocytes. A morphometrical analysis showed that there was no significant difference in the mean size between lactoferrin-positive and myeloperoxidase-negative granules. In contrast, the mean size of myeloperoxidase-positive granules was significantly larger than that of lactoferrin-positive granules. This indicates that lactoferrin is contained in the myeloperoxidase-negative, secondary, granules of human neutrophils. In immature bone marrow mononuclear neutrophils, lactoferrin was present in cytoplasmic granules of somewhat larger size than lactoferrin-positive granules of polymorphonuclear leucocytes. A morphometrical study showed that the mean size of lactoferrin-positive granules was significantly greater in immature bone marrow cells than in polymorphonuclear leucocytes. This indicates that lactoferrin-positive granules decrease in size as the cells mature. Besides cytoplasmic granules, lactoferrin was demonstrated in the Golgi complex and a part of the rough endoplasmic reticulum of immature bone marrow neutrophils, probably myelocytes and early metamyelocytes. These results show that lactoferrin is synthesized and packed into secondary granules in immature bone marrow neutrophils and therefore that the secondary granules are a type of secretory granule.     

Antibody formation in mouse bone marrow. II. Evidence for a memory-dependent phenomenon

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity in a primary response to sheep red blood cells (SRBC), while PFC activity in the secondary response to SRBC can be clearly demonstrated. This phenomenon was studied by means of cell transfer experiments.T cells, which are involved in an anti-SRBC PFC response, were shown to be very scarce in normal mouse bone marrow. This is considered to be the cause of the low PFC activity in the marrow during the primary response to SRBC.In normal mouse bone marrow precursors of IgM-PFC but not of IgG- and IgA-PFC could be found. Priming with SRBC induced the appearance of IgM-, IgG-, IgA- and T-memory cells in the marrow. These B- and T-memory cells were shown to be specific for the antigen which induced their appearance. It is thought that after a second injection of SRBC the IgM-, IgG- and IgA-memory cells can differentiate with the help of the T-memory cells within the bone marrow into IgM-, IgG- and IgA-PFC respectively.The sequence of appearance of the B-memory cells in the bone marrow was shown to be IgM-IgG-IgA.Six months after the intravenous injection of SRBC, the presence of B-memory cells could be demonstrated not only in spleen and bone marrow, but also in peripheral lymph nodes, mesenteric lymph node, Peyer's patches, thymus and blood. The increase in amount of B-memory cells was most prominent in the spleen.     

Ultrastructure of the bone marrow of the salamander Plethodon glutinosus (Caudata: Plethodontidae)

The unusual lymphogranulopoietic bone marrow of the large lungless salamander Plethodon glutinosus was examined by light and electron microscopy. Developing neutrophils, eosinophils, and fat cells were found in large numbers, while lymphocytes of various sizes, plasma cells, plasmablasts, macrophages, pigment cells, and fibroblasts were present in more moderate numbers. Basophils were observed only rarely. Macrophages were found in extravascular locations and did not appear to be associated directly with the walls of the blood vessels supplying the marrow. Both neutrophils and eosinophils seemed to arise from small precursor cells whose ultrastructural features bore a resemblance in some ways to those of mammalian myeloblasts described by Bainton and Farquhar ('66). Developing neutrophils and eosinophils seemed to produce only single populations of specific cytoplasmic granules, rather than both primary (azurophilic) and secondary (specific) inclusions, as are produced typically by mammalian granulocytes. Both eosinophilic and neutrophilic granules were formed in association with Golgi complexes; and eosinophilic granules were much larger, more densely stained, and more regularly rounded in shape than the inclusions of developing neutrophils. Peroxidase activity was associated with the specific granules of neutrophils but seemed to be lacking in the granules of eosinophils. The specific granules of eosinophils were especially unusual because they contained irregularly shaped, lightly stained cores which occasionally displayed a distinctly crystalline substructural organization. The specific granules of basophils also possessed a prominent crystalline organization. The overall appearance of the marrow of Plethodon suggests that it functions not only as a valuable source of neutrophils, eosinophils, and cells of the lymphoid series, but also as a part of the phagocytic system of the animals and as an important repository for fat.     

Immunogold localisation of ubiquitin-protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils.

Ubiquitin-protein conjugates are found in the primary (azurophilic) lysosome-related granules but not in the secondary (specific) granules in mature polymorphonuclear neutrophils prepared from bone marrow. This is the first reported demonstration of ubiquitin-protein conjugates in lysosome-related membrane-bound vesicles in granulocytes and complements our previous findings of ubiquitinated proteins in lysosomes of fibroblasts. The significance of the selective presence of conjugates in only one of the two main types of neutrophil granules remains to be elucidated but may relate to the presence of the complement of acid hydrolases, including proteases, in the azurophilic granules compared to the specific granules. Ubiquitin-protein conjugates may enter the primary granules during neutrophil maturation by an autophagic process or by a heterophagic process during the fusion of phagosomes with primary granules. Alternatively protein ubiquitination may be involved in granule biogenesis.