Special techniques in diagnostic electron microscopy.

The power of electron microscopy as a diagnostic tool can be amplified considerably by the application of ancillary preparative and analytic methods. Subcellular chemistry and structure can be examined by various forms of microprobe analysis and by special staining methods, including cytochemical, immunocytochemical, and negative staining. Qualitative ultrastructural examination can be augmented by morphometric analysis. Correlative microscopic survey methods can be used as a means of targeting ultrastructural investigations. This article provides an overview of the use of these special techniques in the diagnosis and classification of tumors and other selected pathologic processes.

Histology of conjunctival vascular endothelium after filtering surgery with mitomycin C in rabbits.

OBJECTIVE: To examine histologic changes in conjunctival vasculature during the first 72 hours following filtering surgery with adjunctive mitomycin C in rabbits. DESIGN: Thirty-six New Zealand white rabbits underwent unilateral posterior lip sclerectomy. In 18 rabbits mitomycin C (0.5 mg/mL) was applied subconjunctivally for 5 minutes. The remaining 18 animals were treated with the phosphate-buffered saline vehicle for 5 minutes. Three rabbits from either group were killed at 0, 4, 12, 24, 48 and 72 hours postoperatively. OUTCOME MEASURES: Degree of vascularity of filtering blebs on gross examination and appearance of conjunctival vascular specimens on light and transmission electron microscopy, as assessed by three masked observers. RESULTS: On gross examination the filtering blebs in the mitomycin C group were slightly less hyperemic than those in the control group at 12, 24 and 48 hours and were markedly less hyperemic at 72 hours. Light microscopy showed tightly packed erythrocytes within the conjunctival vessels of the experimental blebs and an almost total absence of endothelium within many of the vascular channels by 72 hours. Transmission electron microscopy showed focal loss of vascular endothelial cells and thrombus formation in the experimental blebs, as early as 12 hours postoperatively. CONCLUSIONS: Our findings suggest that the decreased vascularity of filtering blebs observed after trabeculectomy with adjunctive mitomycin C is at least partially due to a toxic effect of the agent on the endothelial cells of the conjunctival vessels.

Filamentous inclusions in acinar cell carcinoma of the pancreas.

Acinar cell carcinoma of the pancreas exhibits a spectrum of histologic appearances. Some tumors can be readily identified by light microscopy, but others resemble endocrine/neuroendocrine neoplasms. Ultrastructurally, though large zymogen granules of acinar cells are usually distinctive, the zymogen granules of neoplastic acinar cells are sometimes abnormally small, overlapping in size with the granules of endocrine/neuroendocrine neoplasms. Six cases of acinar cell carcinoma, two with a typical histologic appearance and four that resembled endocrine/neuroendocrine tumors, were studied ultrastructurally. In addition to zymogen granules and abundant rough endoplasmic reticulum, all cases of acinar cell carcinoma exhibited pleomorphic, membrane bound inclusions that contained filaments. Similar inclusions were not identified in islet cell or carcinoid tumors, and several findings indicate that the inclusions represent deranged zymogen granules. In the ultrastructural study of a pancreatic neoplasm with granules, these inclusions may provide a clue for the diagnosis of acinar cell carcinoma.

Non-heme mechanisms for T1 shortening: pathologic, CT, and MR elucidation.

PURPOSE: To further elucidate the nonparamagnetic effects of T1-relaxation mechanisms in MR imaging. PATIENTS AND METHODS: In 12 patients with lesions having hyperintense signal on T1-weighted spin-echo MR, findings were correlated with autopsy/surgical biopsy in seven cases and/or noncontrast CT scans in 10 cases. RESULTS: Eight of the 10 CT scans demonstrated hyperattenuation in the lesions, indicating mineralization, which correlated with the areas of hyperintense signal on MR. Histologic characterization of the mineralization was accomplished in three cases using four stains; hematoxylineosin, alizarin red S, von Kassa stains for calcium and Perls' iron. The areas of mineralization were homogeneously strongly positive with the calcium stains and only focally weakly positive with the Perls' iron stain. The mineralization was further characterized in all three cases as containing calcium and phosphorus using energy-dispersive x-ray analysis. Four of the 12 cases had either no correlating CT scans (two cases) or the CT showed no hyperattenuating properties to the lesions (two cases). In all four of these cases, microscopic examination showed that the gyriform configuration of the cortical hyperintense signal on T1-weighted images correlated with linear zones of nonhemorrhagic laminar necrosis (cerebral infarction). No mineralization, except for an occasional ferruginated neuron, could be demonstrated with the four histologic stains. Specimen MR imaging of formalin-fixed brain sections in one case demonstrated in vitro the gyriform hyperintense signal seen in vivo. CONCLUSION: Our studies describe and pathologically characterize two associations with T1 shortening in neuroimaging unrelated to the presence of heme: 1) calcification and 2) laminar necrosis in cerebral infarction.

Intercellular junctions and tumor behavior in lung cancer.

We examined the ultrastructure of 76 lung cancers and counted the number of intercellular junctions per area of tissue section. The total number of desmosomes, intermediate junctions, and tight junctions ranged from zero to 300 per 0.157 cm2 of tissue section area, and each type of carcinoma showed a considerable range in concentration of intercellular junctions. Multivariate analysis showed that the number of junctions was significantly tied to the probability of the patient's having extensive stage and to survival time, even after accounting for the routine light microscopic diagnosis of small cell versus non-small cell carcinoma. These results suggest that a tumor's ability to spread and metastasize is inversely related to its number of intercellular junctions, and this conclusion appears to apply to both small cell and non-small cell carcinomas.

Neuroendocrine carcinoma of the oral cavity.

We report the clinical and pathologic features of a small cell neuroendocrine carcinoma arising in the oral cavity of a 63-year-old man. The tumor stained positively with neuron-specific enolase and had dense core granules ultrastructurally. Chemotherapy ordinarily given for pulmonary small cell carcinoma induced a clinical remission. When the tumor recurred 8 mo later, focal squamous differentiation was present within the neuroendocrine carcinoma. The patient died 2.5 yr after presentation.

Ultrastructural morphology of the lung in cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians, with much of the morbidity and most of the mortality related to pulmonary complications. The underlying defect in this disease has yet to be precisely defined, so it is somewhat surprising that a comprehensive study of the ultrastructural morphology of the lung in CF has not heretofore been reported. We used transmission electron microscopy to examine the small airways in 15 patients who had died of CF, and compared the findings with 15 disease controls with non-CF chronic airways disease and 15 patients with normal lung morphology. The lung parenchyma was also examined ultrastructurally in 7 patients with CF, 4 disease controls, and 4 normal lung cases. In addition, the literature regarding the ultrastructural morphology of the large airways in CF was reviewed. Patients with CF showed non-specific ciliary abnormalities, hyperplasia of mucous cells, increased numbers of pulmonary neuroendocrine and indeterminate cells, degeneration and sloughing of epithelial cells, and colonization of bacteria of the mucous layer of the small airways when compared with normal controls. Alveoli showed non-specific injury and regeneration of type II pneumocytes. However, these changes were all similar to those observed in the disease controls. Specifically, no cellular or subcellular ultrastructural abnormality unique to CF was observed. It is probable that the most useful ultrastructural approach to the lung in CF in future studies will involve X-ray microanalytical studies of ionic composition using cryotechniques.

Crystalloids in salivary gland pleomorphic adenomas.

Two types of crystalloids in salivary gland pleomorphic adenomas were studied by light microscopy and electron microscopy. The first type of crystalloid, the previously described tyrosine-rich crystalloid, was identified in three (1.5%) of 205 cases. The crystalloids by light microscopy assumed a radial configuration, resulting in the characteristic petal-shaped morphology. Transmission electron microscopy revealed them to be electron-dense, lobular projections without internal structure. Scanning electron microscopy demonstrated a range of morphology from rounded and intact doughnutlike structures to aggregates of irregular, loosely cohesive plates. The crystalloids were backscatter positive by backscattered electron imaging, and by x-ray microanalysis exhibited prominent calcium, phosphorus, and magnesium peaks that were not present in the adjacent tumor tissue; these three elements may be important in the formation and structure of tyrosine-rich crystalloids. The second type of crystalloid was intraductal and birefringent and was identified in 26 (12.7%) of 205 cases. In 21 of these 26 cases the crystalloids were lost on 10% formaldehyde fixation and paraffin embedding. Histochemical stains and x-ray microanalysis did not reveal a definite chemical composition, but did suggest a predominantly organic nature.

Microprobe analysis of chlorpromazine pigmentation.

We describe the histochemical, ultrastructural, and microanalytical features of a skin biopsy specimen obtained from a patient with chlorpromazine pigmentation. Golden-brown pigment granules were present in the dermis, predominantly in a perivascular arrangement. The granules stained positively with the Fontana-Masson stain for silver-reducing substances and negatively with Perl's stain for iron. Electron microscopy revealed dense inclusion bodies in dermal histiocytes, pericytes, endothelial cells, and Schwann cells, as well as lying free in the extracellular matrix. These "chlorpromazine bodies" were quite dense even in unosmicated, unstained ultrathin sections, indicating that the pigmentation is related, at least in part, to the inclusions. Microprobe analysis of the chlorpromazine bodies revealed a striking peak for sulfur, which strongly suggests the presence of the drug or its metabolite within these inclusions.

The use of a panel of monoclonal antibodies in ultrastructurally characterized small cell carcinomas of the lung.

In recent electron microscopic studies of 51 small cell carcinomas of the lung, the ultrastructural features of epithelial differentiation, particularly the presence of desmosomes, were associated with a tendency toward localized disease, clinical resectability and relatively long survival. Thirty-three of these cases were studied with a panel of monoclonal antibodies (B72.3, B1.1, AE1-AE3 and anti-Leu-7) directed against tumor-associated glycoprotein (TAG-72), carcinoembryonic antigen (CEA), cytokeratin and Leu-7 (an antigen common to natural killer cells and small cell lung carcinomas) to assess the correlation between the immunocytochemical and ultrastructural evidence of differentiation in tumors lacking differentiation at the light microscopic level. Of four small cell carcinomas with ultrastructural glandular differentiation, two were positive for CEA, three were positive for cytokeratin, and none were positive for TAG-72 and Leu-7. Of six tumors with ultrastructural squamous features, cytokeratin was expressed by three, CEA by one and TAG-72 and Leu-7 by none. Of the 23 classic oat cell carcinomas, cytokeratin was expressed by 14, Leu-7 by 3, CEA by 1 and TAG-72 by none. While the pattern of antigen expression did not predictably reflect the submicroscopic features, there was a significant association between keratin staining and extent of disease. The prospective use of similar antibody panels with both cellular and histologic material may therefore help to define clinically relevant categories of this biologically heterogeneous neoplasm.

Quantitative analysis of particulate burden in lung tissue.

Numerous methods have been used in the preparation and analysis of the particulate matter deposited in human lungs. Preparation techniques include those for particle isolation and for in situ analysis. Analytical techniques include bulk and particle-by-particle analysis. In this paper, a general discussion of many of these methods is presented along with examples of how two specific techniques have been used. In one study, individual particles from the lungs of 75 randomly selected autopsy cases were analyzed using an automated scanning electron microscopy (SEM)/energy dispersive X-ray microanalysis (EDX) system. An average of 613 million particles, of exogenous origin, per gram of dry lung tissue were found, the major classes of particles being silica, talc, aluminum silicates, and rutile. In the second study, lungs from 50 randomly selected autopsy cases were analyzed using gravimetric and X-ray diffraction (XRD) analysis. The median total particulate material was 0.33 grams, for cases in which samples were prepared by high temperature ashing, and 0.41 grams, for those in which nitric acid digestion was used. The median amount of quartz for all cases, was 0.044 grams. Samples of eighteen of the 75 lungs previously analyzed by automated SEM/EDX were also analyzed using gravimetric and XRD analysis. A good correlation was seen between the results of the two procedures (r = 0.91 for number of exogenous particles versus grams of particulate matter and r = 0.97 for number of silica particles versus amount of quartz).

Pulmonary carcinomas with a sarcomatoid element: an immunocytochemical and ultrastructural analysis.

Eight primary carcinomas of the lung with a prominent spindle-cell sarcomatoid component were studied by immunocytochemical staining and electron microscopy. The eight tumors were indistinguishable by conventional light microscopy, with the exception of one unusual neoplasm that followed multiple pathways of differentiation with elements of squamous cell carcinoma, rhabdomyosarcoma, chondrosarcoma, and an undifferentiated spindle-cell population. Reticulin fiber production by individual spindle cells and a sharp demarcation of the carcinomatous and sarcomatoid domains by light microscopy were not useful differentiating features. Three of the eight tumors exhibited keratin expression in both the carcinomatous and spindle-cell components. Both immunocytochemical and electron microscopic analyses were required to detect epithelial differentiation, as in one case keratin was identified only by immunocytochemical staining and in another only by ultrastructural examination. Epithelial differentiation was undetectable in the sarcomatoid component of five tumors, and in one case immunoreactive myoglobin was identified in spindle cells; skeletal muscle differentiation was confirmed ultrastructurally. We propose that pulmonary carcinomas exhibiting evidence of epithelial differentiation in a sarcomatoid component be termed spindle-cell carcinomas and that those biphasic tumors exhibiting mesenchymal differentiation into specific tissues, such as neoplastic bone, cartilage, or striated muscle, or lacking epithelial differentiation by light microscopy, immunocytochemistry, and electron microscopy be classified as carcinosarcomas. This distinction may ultimately be unnecessary, because these two tumors may represent different points along a morphologic and biologic continuum.

Subcellular changes in capillary endothelial cells during repair of hyperoxic lung injury.

We studied the changes in subcellular ultrastructure associated with the hypertrophy of capillary endothelial cells during repair of hyperoxic (100% O2) lung injury in rats. We used stereologic-morphometric measurements at different magnifications to determine the absolute volume of each subcellular compartment per average capillary endothelial cell. The increases in this value during the first 3 days of postexposure repair were 118% for cytoplasm, 786% for polyribosomes, 310% for rough endoplasmic reticulum, and 79% for mitochondria; the volume of pinocytotic vesicles did not change. By day 7 of repair, only the polyribosomes and rough endoplasmic reticulum were still increased; by day 14 all values were normal. We conclude that the capillary endothelial cell hypertrophy that develops during repair of hyperoxic lung injury is associated with large and heterogeneous increases in subcellular organelles and is not merely due to increases in the cytosol or to cellular edema. These increases seem to be an integral part of the repair process and may be important in the development of tolerance to subsequent oxygen exposure.

Pleural amyloidosis.

Pleural amyloidosis has been reported rarely, and the diagnosis of this disease by Cope needle biopsy has, to our knowledge, been reported only once previously. We report two patients in whom the diagnosis of pleural amyloidosis was made by biopsy specimens obtained in the examination of large, unilateral pleural effusions. In one patient, the diagnosis was made by Cope needle biopsy. The clinical and diagnostic significance of these cases are discussed.

Polypoid tumor of the esophagus.

Five cases of an uncommon esophageal tumor consisting of a mucosal squamous cell carcinoma that surrounds a polypoid mass of spindle cells were examined. The spindle cell component was composed of elongated cells with blunt nuclei, admixed with multinucleated giant cells. Reticulin fibers enveloped individual cells, and abundant collagen was present. Thirteen to 69 mitotic figures occurred per 10 high-power fields. Electron microscopy showed dilated cisternae of rough endoplasmic reticulum and peripheral intermediate filaments within the cytoplasm. Intermediate-type junctions (zonulae adherens) and subplasmalemmal linear densities connected some cells. No tonofibrillar bundles or desmosomes (maculae adherens) were present. Immunoperoxidase stains detected no keratin in the spindle cells. Alpha-1-antichymotrypsin and alpha-1-antitrypsin were in the spindle cells in five of five and three of five cases, respectively. The absence of desmosomes, tonofibrillar bundles, and keratin and the presence of alpha-1-antitrypsin and alpha-1-antichymotrypsin favor fibrohistiocytic differentiation of the spindle cell component.

Intercellular junctions and tumor stage in small cell carcinoma of the lung.

The authors have studied the ultrastructural features of 52 cases of oat cell carcinoma of the lung and have related their observations to tumor stage and patient survival. Only the type of cell junctions seems to be of prognostic importance. Tumors with intermediate junctions--and especially those with desmosomes--have a more localized stage and may be resectable to result in longer survival than expected for oat cell carcinomas without junctions. For example, in the authors' series the median survival periods for those with no identifiable junctions, intermediate junctions, or desmosomes were 6.4, 8.2, and 11.3 months, respectively. Nevertheless, this ultrastructural subclassification is not as effective as that obtained from careful clinical staging.

Primary brain tumours in Fischer 344 rats chronically exposed to acrylonitrile in their drinking-water.

Acrylonitrile (ACN) has been tested for carcinogenicity by various routes in a number of rat strains. At relatively high levels of administration (e.g. 500 ppm in the drinking-water) there were statistically significant increases in microscopically detectable primary brain tumours, which were difficult to classify. In a further study of ACN-induced brain tumours, ACN was administered to groups of 50 male and 50 female F-344 rats from 6 wk of age at levels of 0, 100 and 500 ppm in the drinking-water. A fourth group of 300 rats (147 males, 153 females), was also given 500 ppm ACN. Neurological signs were observed in 0, 4, 16 and 29, respectively, of the rats in these four groups within 12-18 months. Among the treated animals, females died slightly earlier than males. Few controls of either sex had died by month 18, but, apart from those killed for tumour donation, a high proportion of the rats in the 500-ppm groups had died by that time. Of the 49 brain tumours found in rats exposed to 500 ppm ACN, 11 were only detectable microscopically, 28 were 1-5 mm in diameter and 10 were greater than 5 mm. Despite this variation in size, all the tumours were similar in cellular and architectural features. They were densely cellular, with occasional areas of focal necrosis, and were infiltrative at the margins. They were negative for glial fibrillary acidic protein (GFAP). Ultrastructurally, the tumour cells showed intermingling cytoplasmic processes but no glial filaments and no neurosecretory granules or specialized cell contacts. Samples of tumour tissue were successfully grown in culture, but transplantation of samples from these cultures (observed for up to 12 wk) was unsuccessful. However, a direct intracerebral transplantation from a large tumour was successful.

Sequential changes in lung morphology during the repair of acute oxygen-induced lung injury in adult rats.

We studied changes in lung ultrastructure and collagen content during the repair of acute lung injury in adult rats exposed to 100% O2 for 60 h and recovering in ambient air. In the interstitium, during the first 3 days of repair, the number of neutrophils decreased 16-fold, and monocytes and lymphocytes increased to 7-fold and 4-fold the respective control values. Myofibroblasts increased about 5-fold and the volume of the interstitial matrix remained high. By 7 days, the differential count of inflammatory cells was normal although the number of total interstitial cells and myofibroblasts decreased more slowly. In the capillary endothelium, after 3 days of repair, the cells were hypertrophied and had organelle-rich cytoplasm, and total cell number had increased back to control values; endothelial cell number increased an additional 63% between 3 and 7 days of repair. In the epithelium, type 2 cells increased 150% during the first 3 days of repair before decreasing; type 1 cell number did not change. After 28 days of repair, the lungs appeared qualitatively almost normal; however, interstitial cell number and collagen content were still increased. We conclude that the repair of oxygen-induced lung injury involves a complex pattern of morphologic changes that has important similarities to those occurring during repair on other tissues such as the skin.