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Although Sézary cells are not specific for the Sézary syndrome, the enumeration of these circulating abnormal lymphoid cells is important in the evaluation of patients with mycosis fungoides, Sézary syndrome, and benign dermatoses that might be confused with these entities. Various techniques have been developed for this purpose, but none of the techniques have been widely accepted as practical and accurate. We describe a new method of preparing blood specimens for the identification and enumeration of circulating Sézary cells. Peripheral blood was separated on a density gradient and the mononuclear cell fraction was embedded in plastic. Semithin sections were cut, stained for alpha-naphthyl acetate esterase, and examined by light microscopy. We found that the percentage of Sézary cells correlated well with the clinical diagnosis of Sézary syndrome in the subjects who were studied.
Bone marrow biopsies are now employed in the investigation of many disorders in haematology, oncology and internal medicine. This review provides a survey of the recent literature and a summary of observations made on undecalcified bone marrow biopsies embedded in plastic. The conditions investigated include osteopathies, myelopathies, haematologic and non-haematologic malignancies in the bone marrow. The interrelationship and interdependence of bone and bone marrow have been emphasized, and examples of the effects of diseases of bone on marrow, and of disturbancies of marrow function on bone, have been given. In the myelo- and lympho-proliferative disorders bone marrow biopsies contribute to diagnostic evaluation and classification, as well as to provide factors of prognostic significance. In the investigation of patients with solid tumours bone marrow biopsy may detect metastases in 20 per cent (bronchus), 35 per cent (prostate), 40 per cent (breast), to 80 per cent (unknown primaries) of the patients. Bone marrow biopsy constitutes an additional investigative parameter capable of providing valuable information in many different clinical situations.
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The histochemistry of Hodgkin's cells is controversial. Limited numbers and the variety of techniques used make comparisons difficulty. Acid phosphatase and nonspecific esterase have been the two enzymes most frequently tested because the presence of these enzymes has been thought to be characteristic of histiocytes. Despite the controversial results, these studies have frequently figured prominently in arguments about the cell of origin in Hodgkin's disease. Authors identifying the presence of acid phosphatase and nonspecific esterase have generally favored a mononuclear phagocyte origin; those with negative results have favored a lymphoid origin. We have evaluated a series of 21 cases of Hodgkin's disease using tissues embedded in plastic and tested for acid phosphatase and nonspecific esterase. We found that Hodgkin's cells were frequently positive for acid phosphatase (20 to 21 cases) and/or nonspecific esterase (18 of 21). The reactions are weak and sensitive to inhibition by processing procedures. The reaction patterns are unusual for lymphoid cells and histiocytes. The finding is similar to that seen in interdigitating reticulum cells, a specialized cell found in the paracortex of human lymph nodes.
Previous studies with the light microscope of paraffin-embedded, metal-impregnated sections have probably underestimated the narrow fibre components of the inferior alveolar nerve in the cat. In this study the inferior alveolar nerve of a young adult cat was fixed in situ with glutaraldehyde, post-fixed in osmium and embedded in plastic. A 1 micrometer section of the whole nerve, and an ultrathin section of its largest bundle were cut. Montage micrographs were made from both sections, and from tracings the number and size distribution of the nerve fibres were measured. The entire nerve contained 6856 myelinated nerves whose diameters were distributed bimodally with peaks at 3--4 and 9--10 micrometers. This total is higher than all previously published counts of this nerve, the increase being due to the detection of a greater number of narrower myelinated fibres. In the bundle examined electron microscopically 51.4% of the fibres counted were non-myelinated, four times as many as found in previous studies. The number and distribution of myelinated fibres found by electron microscopy was very similar to that found by light microscopy. The size distribution of myelinated nerves was similar in all the bundles examined. Apart from a lower incidence of non-myelinated fibres, the population of the core of the larger bundle was similar to that of the periphery.
BACKGROUND: The synthesis of Mallory body (MB) and cytoskeletal proteins in hepatocytes have not been studied in vitro. To visualize the incorporation of radiolabeled amino acid into MBs and cytoskeletal filaments separately in the same cell, we developed a new technique using high resolution electron microscopic autoradiography. EXPERIMENTAL DESIGN: Primary cultures of hepatocytes isolated from griseofulvin (GF)-fed mice and control mice were used. The hepatocytes labeled with [35S]methionine were extracted with detergent. The detergent-resistant residual cytoskeleton was characterized by polyacrylamide gel electrophoresis and autoradiography. For light microscopic and electron microscopic autoradiography, the hepatocytes were labeled with [3H]methionine or [3H]leucine, then extracted with detergent and then embedded in plastic. To obtain high resolution of the labeled cytoskeleton, exposed ultrathin sections were treated by the gold latensification and physical developing methods and then examined by electron microscopy. RESULTS: Autoradiography of polyacrylamide gel electrophoresis showed [35S]methionine was incorporated into three major bands: cytokeratin 55, cytokeratin 49, and actin in control and GF hepatocytes. In GF hepatocytes, high molecular weight proteins that remained near the top of the gel (MB-derived proteins) were also labeled. The electron microscopic autoradiography revealed that the intermediate filaments, microfilaments, MBs, and nuclear matrix were labeled and the resolution of the labeling was high enough to localize the labeled amino acids incorporated into the filaments. The background was very low. The labeling using [3H]methionine was greater than [3H]leucine, but the pattern of labeling was the same. In control mice, hepatocytes intermediate filaments and microfilaments were heavily labeled at the cell border. In GF-fed mice, the MBs were heavily labeled compared with the intermediate filaments at the cell borders within the same cells. The intensity of labeling varied from cell to cell. CONCLUSIONS: These results suggested that the newly synthesized cytokeratin monomers were incorporated into the already polymerized filamentous network as well as the MB filaments in cell culture.
Kidneys from broiler chickens at slaughter (6 to 7 weeks of age and mixed sex) were collected by U.S. Department of Agriculture personnel. Forty kidneys were collected from grossly normal birds with grossly normal kidneys (Group D). Grossly swollen kidneys were collected from birds that were otherwise grossly normal (Group A), that had gross lesions of Marek's disease (Group B), or that had gross lesions of squamous cell carcinoma (Group C). These kidneys were fixed in Carson's 10% buffered formalin, embedded in plastic, sectioned at 2 microns, and then stained with periodic acid-Schiff-hematoxylin for glomerular cell counting. Tissue was also processed for electron microscopy from six Group D birds and from six birds total from Groups A, B, and C that had the most severe histologic glomerular lesions. Glomerular tuft cell counts and visceral epithelial cell counts were performed by light microscopy at a magnification of 100x (oil immersion). Total tuft cell counts of all abnormal groups (A, B, and C) were significantly higher (P < 0.05) than those of Group D. Reptilian glomerular tuft cell counts for all abnormal groups were significantly higher (P < 0.05) than the reptilian glomerular tuft counts for Group D. Mammalian glomerular tuft cell counts for Groups A and B were significantly higher than those of the control group (Group D). Electron microscopic examination of the glomeruli from selected birds revealed no dense deposits in the basement membrane and no effacement of epithelial podocyte foot processes. No essential ultrastructural differences were noted between the control group and the abnormal groups.
Microwave fixation methods are important because excellent preservation of both cell structure and antigenicity can be attained several orders of magnitude faster than by routine chemical fixation methods. Fast and ultrafast microwave fixation have yielded significant logistic advantages over another fast fixation approach-rapid freezing at liquid helium temperatures. For example, specimens used for microwave fixation can be as large as 1 cm3 and cells can remain in suspension. We review in detail both qualitative and quantitative morphologic results obtained by using microwave fixation in sample preparation. We provide tables of biological molecules that are preserved in a variety of human and animal tissues by various microwave fixation methods for histochemistry, immunohistochemistry, cytochemistry, immunocytochemistry, and affinity labelling studies. Limitations of large cavity (e.g., household) microwave ovens often result in irreproducible fixation results. We present calibration and standardization protocols for microwave fixation in large cavity microwave ovens that emphasize a) localization of oven hot spots (i.e., high power) using a neon bulb array, b) magnetron warm-up, c) the use of a water load, d) the use of an agar-saline-Giemsa model to predict the uniformity of irradiation in small samples, e) the use of specimen containers with one dimension less than 1.5 cm, and f) fast specimen handling to prevent conductive heating artifacts after irradiation. Although microwave ovens are commonplace their unique applications in the laboratory environment require special safety considerations, which are reviewed. Advances in microwave technology are providing new means to study the structure-function relationships of cellular and biochemical activities.
This study developed methodology to evaluate the healing of 15 IMZ implants placed in the sockets of freshly extracted mandibular premolars in three adult mongrel dogs. Six surgical sites were prepared in each animal and one site was left as a control. Porous hydroxyapatite was placed around the top half of two implants in each animal; one implant was also covered with polytetrafluoroethylene membrane. All implants were covered with a mucoperiosteal flap and sutured closed. Upon animal sacrifice, the mandibles were retrieved for block dissection and the blocks were embedded in plastic. Serial longitudinal wafers were ground to 50 to 100 microns and stained. Standard photomicrographs were taken so that tracings of the implant-bone interface could be measured on a sonic digitizer. The mean percent amount of bone to plasma-sprayed portion of the implant on the longitudinal sections was 47.9% +/- 5.2% with a range of 17.4% to 84.7%. The horizontal sections were measured at 53.5% +/- 3.7% with a range of 0.0% to 100%. This pilot study suggests that IMZ implants placed in fresh extraction sockets can achieve a degree of intimate contact with bone; however, wide variation in the implant-to-bone interface was found even in the same specimen.
The fine structure of the retinal epithelium (RPE), choriocapillaries and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the great horned owl (Bubo virginianus). The RPE consists of a single layer of cuboidal cells joined laterally in the mid to basal region by a series of tight junctions forming part of the blood-ocular barrier. Basally (sclerally) the epithelial cells show numerous deep infoldings while apically (vitreally) a wealth of microvillar processes interdigitate with the photoreceptor cells. Internally the RPE cells display a large vesicular nucleus, plentiful smooth endoplasmic reticulum (SER) and polysomes with only small scattered profiles of rough endoplasmic reticulum (RER). Numerous pleomorphic mitochondria are basally located. In the light-adapted state the melanosomes are located almost exclusively within the apical processes indicating retinomotor movements. Myeloid bodies are numerous and often show ribosomes on their outer surface. Bruch's membrane is typical of avian species in that it is pentalaminate and the lamina densa is displaced towards the choriocapillaris. The choriocapillaris itself is but minimally fenestrated facing Bruch's membrane. Most fenestrations present show a single layered diaphragm while others display a double-layered diaphragm.
The retinal photoreceptors of the great horned owl (Bubo virginianus) consist of rods, single cones and unequal double cones present in a ratio of about 30:1.2. In the light-adapted state the rods are stout cells which are not felt to undergo retinomotor movements. The rod outer segment consists of a stack of scalloped membranous discs enclosed by the cell membrane. The rod inner segment shows an ellipsoid of mitochondria and a wealth of rough endoplasmic reticulum (RER) and polysomes, Golgi zones and autophagic vacuoles but not hyperboloid of glycogen. Single cones show a slightly tapered outer segment, a heterogenous oil droplet and an ellipsoid of mitochondria at the apex of the inner segment. Double cones consist of a larger chief member which also displays an oil droplet and a slightly smaller accessory member which does not. Both members of the double cone as well as the single cone show a prominent ellipsoid, plentiful polysomes and RER and Golgi zones in the inner segment. Neither single nor double cones possess a condensed paraboloid of glycogen but instead show plentiful scattered glycogen particles. Along the contiguous membranes between accessory and chief cones a few presumed junctional complexes are seen near the external limiting membrane. Judging by their morphology in light-adaptation the cones of this species do not undergo photomechanical movements. Rods and cones (both types) have both invaginated (ribbon) and numerous superficial (conventional) synaptic sites. Rods are more numerous in this nocturnally active bird than is usually noted in avian species.
The pecten oculi of the great horned owl (Bubo virginianus) has been examined by light and electron microscopy. The pecten in this species is of the pleated type and is small in comparison to the size of the eyeball. It consists of 7-8 accordion folds which are joined apically by a pigmented bridge of tissue. Within each fold are numerous capillaries, larger supply and drainage vessels and plentiful pleomorphic melanocytes. The capillaries are extremely specialized vessels, most of which display plentiful microfolds on both their luminal and abluminal surfaces although some capillaries show but a few microfolds. The endothelial cell bodies are extremely thin with most organelles located near the nucleus. All capillaries are surrounded by a thick fibrillar basal lamina which is felt to be structurally important. Pericytes are a common feature within these thickened basal laminae. The numerous melanocytes form an incomplete sheath around the capillaries and are also presumed to be fulfilling a structural role. While the morphology of the pecten in the great horned owl is certainly indicative of a heavy involvement in transport, when compared to the pecten in species that are more visually oriented it is smaller, displays fewer folds and a reduced number of microfolds within the capillaries.
The present investigation has re-examined the hypothesis proposing that glycoprotein (GP)Ib/IX receptors for von Willebrand factor are rapidly cleared from exposed surfaces to internal membrane systems after activation of platelets by thrombin in suspension. Platelets were prelabeled with either a polyclonal antibody to GPIb alpha, antiglycocalicin (A-Gl), or a cocktail of two monoclonal antibodies, AP1 and 6D1, exposed to 0.1 or 0.2 U/ml thrombin for 5 or 10 minutes, fixed and stained with Staphylococcus protein A coupled to gold to detect A-Gl or goat anti-mouse IgG bound to gold particles to locate AP1 and 6D1 before or after preparation of frozen thin sections or embedding for plastic thin sections. The frequency and distribution of protein-A-gold markers for GPIb/IX on thrombin-activated platelets viewed in thin plastic sections did not differ from the density on resting platelets stained with A-Gl. Cryosections of A-Gl-prelabeled platelets labeled again on cryosections revealed GPIb present on linings of the open canalicular system of resting and activated platelets, but the density of gold in interior channels and frequency of gold particles on exterior surfaces were not altered by thrombin stimulation. Platelets prelabeled with the cocktail of 6D1 and AP1 and studied in cryosections also failed to reveal uptake of GPIb/IX receptors into the open canalicular system after activation by thrombin. The findings do not support the concept that thrombin causes clearance of GPIb/IX receptors from exterior surfaces to interior membranes of activated platelets.
The retinal pigment epithelium (RPE) as well as the choriocapillaris and Bruch's membrane (complexus basalis) have been studied by light and electron microscopy in the barred owl (Strix varia). The RPE consists of a single layer of cuboidal cells joined laterally by a series of tight junctions that forms part of the blood-ocular barrier. Basally (sclerally) the retinal epithelial cells display numerous deep infoldings while apically (vitreally) microvillar processes interdigitate with the photoreceptor outer segments. Internally the RPE cells show a large vesicular nucleus, plentiful smooth endoplasmic reticulum (SER) and polysomes but very little rough endoplasmic reticulum (RER). Numerous pleomorphic (including ring-shaped) mitochondria are basally located. In the light-adapted state the small melanosomes are almost exclusively located within the apical process indicating that retinomotor movements probably occur. Phagosomes and lysosome-like bodies are present as are myeloid bodies which may show ribosomes on their outer surface. Bruch's membrane is typical of avian species in that it is pentalaminate and the central lamina densa is displaced towards the choroid. The choriocapillaris endothelium is thin but only minimally fenestrated facing Bruch's membrane. Most fenestrations present show a single-layered diaphragm while others display a double-layered diaphragm as noted in other avian species.
The photoreceptors of the barred owl (Strix varia) consist of rods, single cones and unequal double cones present in a ratio of about 35:1:3. In the light-adapted condition the rods are of uniform diameter along their entire length and are therefore not felt to undergo photomechanical changes. The rod outer segment consists of a stack of scalloped bimembranous discs enclosed in a limiting membrane. The rod inner segment displays an ellipsoid of mitochondria, much rough endoplasmic reticulum (RER), numerous polysomes, Golgi zones and autophagic vacuoles, but no hyperboloid of glycogen. Single cones show a slightly tapered outer segment and a heterogeneous oil droplet along with an ellipsoid of mitochondria at the apex of the inner segment. Double cones consist of a larger chief member which also displays a heterogeneous oil droplet and a slightly smaller accessory member which does not. Both members of the double cone as well as the single cones show plentiful polysomes and RER as well as Golgi zones in the inner segment, but none of the cones possessed a condensed paraboloid of glycogen. The contiguous membranes of the chief and accessory cones displayed a few presumed junctional complexes. Judging by their elongated shape in the light-adapted state, cones in this species do not undergo retinomotor movements. Rods and both types of cones have both invaginated (ribbon) and superficial (conventional) synaptic sites.