BELL, N.,TUCKER, J.A., EVANKOVICH, C., VU, H., LATOUR, L.F., SIRMON, M.D., HARDIN, J.G., RODNING, C.B., University of South Alabama, 2451 Fillingim Street, Mobile, Al. 36617. ImDortance of morDholoaic markers of locallv increased interferon levels in the ultrastructural evaluation of renal biomies. Tubuloreticular inclusions (TRI) and cylindrical confronting cisternae (CCC) are structures which can be identified ultrastructurally and have been associated with locally increased interferon levels in tissues. TRI have long been recognized to occur in a variety of conditions, particularly autoimmune diseases. CCC are recognized in a limited number of conditions. The combination of TRI and CCC have only been detected in a few disease states including human immunodeficiency virus infection, multiple sclerosis, adult T-cell leukemia, systemic lupus erythematosus, some neoplasms, and patients undergoing interferon treatment. As such, these structures can be useful in narrowing a differential diagnosis.

A 36 year old man underwent splenectomy for the treatment of hereditary spherocytosis. Postoperatively, he rapidly developed renal failure, and he underwent renal biopsy to evaluate this problem. By light microscopy, the kidney exhibited numerous glomerular crescents, and the category of rapidly progressive glomerulonephritis seemed likely. Ultrastructurally, however, deposits were seen, and both TRI and CCC were identified. These findings suggested the alternative possibility of lupus nephritis. Additional laboratory study in conjunction with his clinical findings confirmed this diagnosis. In this case, the identification of TRI and CCC was important for the proper categorization and treatment of this patient’s disease.

HISTOLOGIC AND ULTRASTRUCTURAL ANALYSIS OF PHEOCHROMOCYTOMAS. J. Elliot Carter, M.D., Judy A.C. King, M.D., Ph.D., and J. Allan Tucker, M.D., Department of Pathology, University of South Alabama, Mobile, AL.

Recently, a twenty-six year old female presented to our hospital and was diagnosed with multiple endocrine neoplasia syndrome, a feature of which is the presence of pheochromocytomas of the adrenal glands. The adrenal glands are paired endocrine organs located in the retroperitoneum superomedial to the kidneys. They are a composite of two types of endocrine tissue, one mesodermally derived (cortex) and the other neuroectodermally derived (medulla). The predominant cell type in the medulla of the adrenal gland is the pheochromocyte (medullary or chromaffin cell) which is responsible for the production of epinephrine and norepinephrine. Pheochromocytes are large polygonal cells with poorly outlined borders and abundant, distinctly granular cytoplasm. Ultrastructurally, the pheochromocytes can be divided into two types on the basis of the type of cytoplasmic dense-core neurosecretory granules they contain: norepinephrine containing (electron opaque, often eccentrically located within a dilated sac, approximately 250 nm in diameter) and epinephrine containing (finely granular, filling the enclosing membrane, about 190 nm in diameter).

Neoplasms of the pheochromocytes are termed pheochromocytomas, and they may secrete epinephrine, norepinephrine, or both. Grossly, they are encapsulated, soft, yellow to red masses which range in weight from a few grams to over 2000 grams. They often have areas of necrosis, hemorrhage, and cyst formation. Microscopically, the tumor cells vary in size and shape and have prominent nucleoli, but have the typical basophilic pheochromocyte cytoplasm. Ultrastructurally, the tumor cells recapitulate the cytoplasmic secretory granules of the non-neoplastic pheochromocytes. The characteristic gross, histologic, and ultrastructural findings of the pheochromocytomas in our patient will be reviewed.

Proceedings of the Sixteenth AnnualMeeting of the Alabama Imaging andMicroscopy Society, Held inBirmingham, AlabamaApril 17–18, 1997

MICROSCOPY RESEARCH AND TECHNIQUE 40:81–84 (1998)

r 1998 WILEY-LISS, INC.

Transmission Electron Microscopic Diagnosis of Amiodarone Pulmonary Toxicity. A. Hafeez Diwan, M.D., and J. Allan Tucker, M.D., Department of Pathology, University of South Alabama, Mobile, AL.

Amiodarone is an oral anti-arrhythmic drug used in long- term therapy of patients with cardiac arrhythmias. Toxic effects include pulmonary toxicity in 2 to 15% of patients, manifested clinically as dyspnea and dry cough. Transmission electron microscopy (TEM) is useful in establishing the diagnosis of amiodarone toxicity in the lung.

We present the case of a 46 year-old white male treated with amiodarone who underwent an open lung biopsy to evaluate the patient for possible amiodarone toxicity. Light microscopic examination of hematoxylin and eosin stained paraffin- embedded sections revealed an abundance of clumped macrophages in alveoli, type II alveolar cell hyperplasia, and patchy mild interstitial fibrosis. On TEM, numerous alveolar marcophages and large type II alveolar cells containing abnormally large and sometimes complex lamellar bodies were seen. While such lamellar bodies within type II alveolar cells are morphologically indistinguishable from surfactant lamellar bodies normally seen, the presence of these within alveolar macrophages is abnormal and consistent with the reported effect of amiodarone in the lung. Thus, the clinical history of amiodarone intake along with these TEM findings help in identifying and diagnosing amiodarone induced pulmonary toxicity.

FAYWICK, D. W., Quinn, L. C. and Stapleton, C., Department of Geology and Geography, University of South Alabama, Mobile, AL 36688. Dissolution of a clam shell (as revealed by thin-section petrography and electron microscopy) Aragonite diagenesis has long been recognized as the primary mechanism in the cementation of most carbonate sediments. Dissolution of aragonite, occurring simultaneously with precipitation of calcite cement, will result in a limestone containing molds and recrystallized (neomorphic) allochems. Compared to the cementation mechanism, processes of initial aragonite dissolution and diagenesis are poorly understood. Aragonite can begin to alter almost immediately in contact with meteoric water. The result is a powdery or “chalky” appearance to shells and skeletal allochems. Our study is focusing on the initial and early diagenetic phases of alteration within aragonitic bivalves. To date, we have examined specimens, primarily of the Genus Chione, from New Zealand (modem and Tertiary Petane Group), south-westem Florida (modem and Tertiary Tamiami Formation), and coastal Alabama (modem). In addition to traditional methods of petrography and geochemical analysis (e.g., light and electron microscopy, electron microprobe, ICPS, stable isotope geochemistry), we have initiated a laboratory-based experiment simulating meteoric diagenesis under controlled settings. Our experiment consists of two water filtration canisters, each containing 10 to 20 weighed portions of Tertiary and modern bivalves, packed in either carbonate sand (mean grain size 250 pm), or siliciclastic sand (mean grain size 125 pm), through which meteoric water passes (flow rate 2 to 4 liday). A third canister containing shells in siliciclastic sand with saltwater-flow acts as a control.

Our results suggest that there is rapid mass loss of aragonite through dissolution during contact with meteoric water and that rates of dissolution may he controlled by shell surface area and fabric. Dissolution initially occurs along outer extremities of shells, hut soon spreads into the interior by way of foliations and other fabric-related planes of weakness. From here, dissolution passes into intracrystalline spaces between the aragonite crystallites that comprise individual layers of shells. In addition to the increase of intracrystalline porosity, initial stages of aragonite diagenesis may be accompanied by net losses in bulk Si‘ and 0”.

ASBESTOS: APPLICATIONS OF ELECTRON MICROSCOPY AND ENERGY-DISPERSIVE X-RAY ANALYSIS. Judy A.C. King, M.D., Ph.D., Song W. Wong, M.D., and J. Allan Tucker, M.D., Department of Pathology, University of South Alabama, Mobile, AL.

The term “asbestos” is derived from a Greek word meaning “inextinguishable” or “unquenchable.” Because asbestos is noncombustible, nonconducting, and chemically resistant, it was used in many products prior to 1971, when the Occupational Safety and Health Administration regulated its use. Mesothelioma and bronchogenic carcinoma have been associated with asbestos exposure. Many people are now interested in occupational exposures that occurred in previous decades. Lists of jobs with estimates of probability of asbestos exposure have been established.

Histologic diagnosis of asbestosis requires both the presence of fibrosis in the walls of respiratory bronchioles and the accumulation of asbestos bodies. When asbestos fibers or other particles become trapped in the lung, they become coated with iron and protein. The structures are called “ferruginous bodies” which means “iron-rich.” Ferruginous bodies can be identified by light microscopy in histologic sections of the lung or in digestion (sodium hypochlorite) specimens of lung.

Although presumptive diagnosis of asbestos bodies can be made when ferruginous bodies with colorless transparent cores of appropriate length are identified by light microscopy, definitive diagnosis requires analysis of individual fibers by scanning electron microscopy and energy-dispersive x-ray analysis. Uncoated fibers greatly outnumber the coated fibers in lung tissue. Only coated fibers (“ferruginous bodies”) are visualized by light microscopy, whereas both types of fibers can be examined with scanning electron microscopy. Energy-dispersive x-ray analysis can be used to identify the specific type of asbestos fiber.

BONNIE L. MARTIN, M.D., JUDY A.C. KING, M.D., PH.D., J. ALLAN TUCKER, M.D., and KELLY P. ROVEDA, M.D. Department of Pathology, University of South Alabama, Mobile, Al. 36617. Role of electron microscopy in the evaluation of metastatic neuroendocrine neoolasms of the abdomen. The endocrine cells of the pancreas, which are predominantly organized into the islets of Langerhans, can give rise to neoplasms designated as islet cell tumors. Endocrine cells in the stomach and bowel can also give rise to neoplasms; these are termed carcinoid tumors. At their respective primary sites, categorization of these tumors is usually straightforward. Both of these lesions, however, have the ability to metastasize. At metastatic sites, these two tumors are generally histologically indistinguishable. Immunohistochemistry and electron microscopy can be useful in further evaluation of these neoplasms.

As an example, a 44 year old male with a history of peptic ulcer disease presented with marked gastrointestinal bleeding, necessitating emergency surgery. A segment of duodenum with a deep ulcer was removed, and the antrum of the stomach was removed to control production of gastric acid. Intraoperatively, peripancreatic masses measuring 1.8 and 3.0 cm were identified. Histologically, these consisted of lymph nodes which were extensively replaced by nests of monotonous, bland cells indicative of a neuroendocrine neoplasm. To evaluate whether the lesions represented metastatic carcinoid tumor or metastatic islet cell tumor, immunoperoxidase stains were performed; stains for somatostatin and insulin were negative, while gastrin yielded possible but inconclusive stainmg. Ultrashuctural evaluation revealed numerous, round granules indicative of an islet cell tumor and not a midgut carcinoid tumor. Though not specific, the granules varied in size and sometimes in density, findings compatible with a gastrin- producing islet cell tumor (gastrinoma).

Although gastrin-producing cells are not found in the normal pancreas, gashinomas do arise in the pancreas and are believed to be derived from primitive, multipotential endocrine cells of the pancreas which have differentiated inappropriately. The peripancreatic location of the lymph nodes and the presentation with severe peptic ulcer disease are in keeping with the gastric hypersecretion associated with gashinomas termed Zollinger-Ellison syndrome.

82 ALABAMA IMAGING AND MICROSCOPY SOCIETY ABSTRACTS

VISCOSITY OF EMBEDDING MEDIA, IMPORTANT OR NOT! JosC A. Mascorro and Gerald S. Kirby, Department of Anatomy, Tulane University School of Medicine, New Orleans, LA.

Embedding media "kits" contain one epoxy resin along with the anhydrides DDSA and NMA and the amine accelerator DMP-30. These packages produce good results, but the final medium will exhibit a constant viscosity that cannot be changed (assuming the researcher wished to do so) because it is specific to the make-up of that particular "kit". However, the viscosity and hardening rate of any embedding medium can be easily manipulated by substituting lower viscosity ingredients that would increase fluidity and slow hardening. For example, DDSA and DMP-30 in the traditional kit will add significantly to overall viscosity and could be replaced by more favorable components, such as NSA and BDMA, in order to produce a less viscous, more manageable, yet equally successful mix.

The high viscosity resins Epon 812 and Eponate 12 were combined with DDSA, NMA, and DMP-30 and tested @ 5, 30, and 60 min for Flow Rate (FR), Volume Flow Rate (VFR) and Viscosity (centipoise/cp). The resins next were prepared in novel combination using NSA (in place of DDSA) and BDMA (in place of DMP-30) and tested similarly. Both resins, when combined with the kit components, showed a slow FR, a low VFR, and a high cp. In fact, the FR for the traditional Epon mix nearly quadrupled by 60 min (from 6 min to more than 23 min), while the VFR was substantially less (mixture slowed from 0.0232 ml/sec to 0.0064 ml/sec). The resulting cp of the mixture more than tripled @ 60 min, from 106.3 to 386.6. When DDSA and DMP-30 were replaced by the NSA and BDMA counterparts, the cp was reduced from a high of 386.6 to a more manageable 167.0. This similar trend was apparent for Eponate and other combinations tested.

These tests suggest that microscopists need not depend solely on kit- arranged embedding media. Viscosity and Hardening Rate can be tempered simply by changing "kit" components, thus producing fluid media that would satisfy the specific needs of a particular tissue type.

NICOLAS David, P., NASA Marshall Space Flight Center, MS EB13, MSFC, AL 35812. Export Laws and Regulations Applied to Scientific Publications and Presentations. There are two laws, ITAR (22 CFR 120) and EAR (15 CFR 730), which control the transfer or export of technology and information to foreign governments and their citizens. The definition of an export is quite general and covers the transfer of anything to a foreign person by any means, anywhere, or anytime. The International Traffic in Arms Regulation covers the hardware and technology associated with missile systems and spacecraft. The Export Administration Regulations are administered by the Department of Commerce and cover ten categories of controlled items including many common technologies: nuclear materials and facilities; chemicals and microorganisms; materials processing; electronics; computers, telecommunications and information security; lasers and sensors; navigation and avionics; marine; and space systems. Scientists and engineers are generally unaware of these restrictions and can easily place themselves in harm's way subjecting themselves to stiff penalties and fines. Both laws have teeth and carry severe criminal and civil penalties with fines ranging up to $1 million per violation and imprisonment of 10 years per violation.

SHARDO, J.D. Department of Biological Sciences, University of South Alabama, Mobile, AL 36688. A morphological description of late embryonic and early larval development of Atlantic salmon, Salmo salar. Modern bony fishes, the teleosts, are the largest and most diverse group of extant vertebrates, comprising over 20,000 species. The general pattern of teleost development has been described but adaptive modifications contributing to teleost diversity are largely unknown. The teleost, Atlantic salmon, is distinguished by large egg size and a prolonged incubation at low temperatures within a gravel nest. By 44 days postfertilization the embryo has formed basic axial structures and the tail has begun to separate from the yolk mass. Next the head separates from the yolk mass, gill arches are formed and the operculum grows to cover the gill arches. At 85 days the mouth opens and the jaws begin to elongate. Gill filaments begin to form around 119 days. Embryos hatch at 138 days but in nature remain in the nest for another month, growing while living off of stored yolk. In Atlantic salmon, development is slow but continuous despite low incubation temperature. Large amounts of stored yolk provide nutrients for the long embryonic incubation and enable the salmon larva to remain in the nest after hatching, thus avoiding predation as growth continues. The developmental pattern of Atlantic salmon is similar to that of other salmonoid species described in the literature.

SHELDON, M.A., Department of Geology and Geography, University of South Alabama, Mobile, AL 36688; and Rochester Institute of Vertebrate Paleontoloav, Rochester,

I_ - ~~~~~~-~ ~ ~

NY 14526. Microscopic research of Upper Cretaceous foszil bone. Research into the histology of fossil bone is a - growing area of inquiry in vertebrate paleontology. Bone preservation of extinct animals from %he Upper Cretaceous is exceeding precious expectations. Preserved details of ontogeny include growth plate at the distal end which shows a classic progression from resting cartilage to newly formed fibrous bone. Gther preserved features are endochondral and periosteal ossified cartilage, features of Haversian canals, Sharpey's fibers and osteocytes airanged within the matrix. SEM analyses of prenatal mosasaiirs show that details of resting, proliferating, hypertrophled cartilage and early circulatory system are Intact.

appears to present new opportunities for gainink data to better understand evolution, ecol.ogy, and growcL. mechanisms of ancient animals. Among some closely related marine lizzrds, mosasauers, generic identification can be made from histological section. Comparative studies bf

marine afid trrrestrial (both extant and extinct animals), resulted in Lhe Liiscovery oi major differences in ?>OLI&

density. 11, tne various marine environments, distidtt regimes o i gravitational and corque stresses .ire exxted during growth and remodeling of bone. Marine -Jerteti.ate bone architecture can be correlated with life specific portions of. the water. shallow regions of the water column and their bone micro- structure is pachyostotic. Many other whales and do1,;hins inhabit deeper portions of the water column and ;heir bone structure is osteoporotic.

lncorporating the study of fossii bone architectuce

Beluga whales and maneters iiiliitbit

83ALABAMA IMAGING AND MICROSCOPY SOCIETY ABSTRACTS

EXPERIENCE WITH THE HYDROPHOBIC EPOXY RESIN DURCUPANB ACM A S AN EMBEDDING MEDIUM FOR PRO- AND EUCARYOTIC SPECIMENS.

Christian T. K.-H. Stadtliinder

Department of Epidemiology, School of Public Health, University of Alabamaat Birmingham, Birmingham, AL35294

Embedding of biological specimens in resins is necessary to provide support during ultrathin sectioning and to allow visualization of the section with a transmission electron microscope. Numerous embedding media have been described in the literature as being suitable for the ultrastructural preservation of single cells and tissues. The most commonly used media include acrylic resins such as polymethyl methacrylate, Lowicryl, Unicryl, and London Resins, as well as epoxy resins such as Epon, Araldite, and vinyl cyclohexane dioxide (VCD).

The present paper describes the experience in using the four- component hydrophobic epoxy resin Durcupan@ ACM for embedding a variety of biological specimens including tissues from trachea, stomach, and fish gills as well as blood cells and bacteria. It was found that the majority of Durcupan@ resin blocks could be easily cut with an ultramicrotome. Only samples in which the resin was mixed unevenly or the plasticiser was added in a too low concentration, blocks appeared brittle and sections were useless for investigation. Although an exchange medium (e.g., propyleneoxide) was required after the alcohol dehydration, the hydrophobicity of the resin appeared as an advantage in cases when the trapezoid block tip accidentally came in contact with the water in the trough. Furthermore, it was found that the resin had excellent beam stability and allowed satisfactory staining of the biological specimens with uranyl acetate and lead citrate. The resin appeared very useful for embedding fragile specimens such as pleiomorphic bacteria which lack a rigid cell wall as well as cells with surface-exposed structures such as ciliated epithelial cells.

Overall, the resin was a little more difficult to handle (careful mixing is required of all four components), when compared to resins such as LR- White or Unicryl. Also, there was a longer embedding procedure (propyleneoxide step, two-temperature polymerization, and post- polymerization aging). However, this hydrophobic resin was found to give an excellent ultrastructural preservation of biological specimens, which is particularly important to answer questions in basic research.

84 ALABAMA IMAGING AND MICROSCOPY SOCIETY ABSTRACTS