Crystalloidal paraprotein deposits in the cornea: an ultrastructural study of two new cases with tubular crystalloids that contain IgG kappa light chains and IgG gamma heavy chains.

The fine structure and immunoprotein content of the crystalloids are described in two cases of paraproteinemic crystalloidal keretopathy, both of which had clinical features thought by the referring ophthalmologists to be those of atypical lattice-type corneal dystrophy (presumably because of lattice-like lines). Most keratocytes in one case were surrounded by a mantle of densely packed tubular crystalloids. Individual tubules were annular in cross section with mean dimensions as follows: overall diameter, 29.32 nm (SD 1.26); internal diameter (core), 8.53 nm (SD 1.12); wall thickness, 10.39 nm (SD 0.85) (n = 10). Crystalloids were extracellular and found only in the corneal stroma, with none in Bowman's layer or Descemet's membrane. In the second case, the tubules had a similar distribution but formed geometric arrays with no clear relationship to, or envelopment of the keratocytes. The tubules were thin-walled, with mean dimensions as follows: overall diameter, 26.12 nm (SD 1.12); internal diameter (core), 15.46 nm (SD 1.12); wall thickness, 5.33 nm (SD 0) (n = 10). In both cases the tubules were kappa-light chain- and gamma-chain-positive. Laboratory investigations revealed the presence of two IgM-kappa paraproteins and an IgG-kappa paraprotein in the serum of the first patient. The second patient had an IgG-kappa paraproteinemia and bone marrow changes consistent with low-grade non-Hodgkin's lymphoma. These cases emphasize and extend the morphological range of corneal IgG crystalloids; the second case also demonstrates that corneal IgG crystalloids may be an early indicator of un underlying immunoproliferative disease.

Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium.

To optimize the ultrastructural localization of immunoglobulin G in corneal crystalloid deposits, we compared a range of antigen unmasking techniques. A human corneal biopsy specimen was fixed in formalin, post-osmicated, and embedded in epoxy resin for electron microscopy. Thin sections were immunogold-labeled for IgG after treatment with sodium ethoxide or sodium metaperiodate. Sections were also treated by heating them at 95 degrees C while they floated on water, 0.01 M citrate buffer (pH 6.0), or sodium metaperiodate. The treatments were applied separately and combined. After labeling, crystalloids in untreated sections had a probe density of 5 particles/microns2. Crystalloids in sections treated only with sodium ethoxide or sodium metaperiodate had probe densities of 15-20 particles/microns2. Sodium ethoxide combined with heating on water, or citrate buffer, gave probe densities of 140-160 particles/microns2. Sodium metaperiodate combined with heating on citrate buffer gave the highest probe density (195 particles/microns2). Although sodium ethoxide coupled with heating increased probe density, the ethoxide etched the sections and caused unacceptable damage. Treatment with sodium metaperiodate followed by heating on citrate buffer is recommended for antigen unmasking. This combination gave a high probe density and sections remained intact, with good ultrastructural detail.

Glomerular basement membrane thinning in a patient with hematuria and hemoptysis mimicking Goodpasture's syndrome.

Ultrastructural morphometric studies of glomerular basement membrane (GBM) thickness are described in two renal biopsy specimens from a patient who presented with hemoptysis and hematuria mimicking Goodpasture's syndrome. Significant GBM abnormality, with attenuation as the main lesion, identified in a biopsy specimen taken during active clinical disease appeared to have resolved in a second biopsy specimen taken during the recovery phase. There was no evidence of glomerulonephritis. Concurrent lung biopsy studies showed focal alveolar-capillary wall basal lamina changes of uncertain diagnostic significance. These observations suggest the alternative possibilities that GBM attenuation may be either an acquired consequence of systemic disease or may be part of an hitherto unrecognized primary multisystem abnormality of basal lamina affecting, in this case, glomerular and pulmonary laminae, resulting in hematuria and hemoptysis. The morphometric studies in this case indicate that simple-mean measurements of GBM thickness are inadequate alone for the quantitative study of this lamina because significant inter- and intraglomerular membrane variation, if irregularly distributed, can remain undetected.

Paraproteinemic crystalloidal keratopathy: an ultrastructural study of two cases, including immunoelectron microscopy.

The ultrastructural appearances of corneal crystalloidal deposits are described in two patients with an IgG-kappa paraproteinemia of uncertain pathogenesis. The crystalloids in one patient were overwhelmingly intracellular and were found mainly in stromal keratocytes, but also in basal corneal epithelial cells and the limbal vascular endothelium. Four types of crystalloid or immunoprotein-containing granules were recognizable in this case: 1) fibrillary crystalloids with a curvilinear filamentous substructure; 2) angulated geometric crystalloids that often had a linear filamentous substructure and transverse or oblique periodicity; 3) cordlike crystalloids; and 4) lysosomelike granules with amorphous contents. Immunoelectron microscopy demonstrated that all of these structures labeled for kappa-light chains, and rectangular type 2 crystalloids showed approximately a twofold greater concentration of the colloidal gold probe than the type 1 fibrillary crystalloids. The evidence suggested development of the crystalloids within lysosomes, with a progression from the granules containing amorphous material, through fibrillary crystalloids, to the geometric structures. The circumferential distribution of the corneal deposits, as well as the presence of vascular endothelial crystalloids and reduplication of external laminae around limbal blood vessels, suggests that the crystalloids originated predominantly or entirely from the blood, with transport of immunoprotein across damaged limbal microvasculature. The abnormal vasculature may also have contributed to corneal edema, which in turn may have exacerbated corneal opacification. The crystalloidal deposits in the other case were exclusively extracellular; they were located beneath and between corneal basal epithelial cells, and predominantly as a mantle around individual keratocytes. The crystalloids in this case consisted overwhelmingly of thick-walled tubules about 40 nm in diameter that labeled for both kappa-light chains and gamma chains with the colloidal gold immunoprobe. In addition, lucent vesicles within keratocytes were found only in sections labeled for kappa-light chains and were positive. The factors that might contribute to the formation of corneal crystalloidal deposits in immunoproliferative disorders are discussed, and include: 1) an inherent propensity for crystallization of some immunoglobulins or kappa-light chains, perhaps because of abnormal molecular structure; and 2) local factors in the cornea that might promote deposition and crystallization of immunoprotein, such as temperature, pH, the water content, and extracellular matrix components.

Use of tannic acid and silver enhancer to improve staining for electron microscopy and immunogold labeling.

Staining by uranyl acetate and lead citrate (UA-LC) of immunolabeled sections of unfixed glomerular basement membrane (GBM) digests embedded in low-acid glycol methacrylate (LA-GMA) is poor. The following were investigated for their ability to enhance contrast when applied to sections before UA-LC: potassium permanganate, phosphotungstic acid, gold chloride, osmium tetroxide, glutaraldehyde-osmium tetroxide, colloidal gold-silver enhancer, tannic acid, and glutaraldehyde-tannic acid. Silver enhancer (2 min incubation, no sodium thiosulfate step) gave dense GBM staining but not with immunogold labeling. Silver enhancer is recommended as a simple alternative to routine silver stains but not for contrast enhancement with immunogold labeling. Tannic acid (1% for 1 min or 0.005% for 10 sec) and glutaraldehyde (2.5% for 5 min) followed by tannic acid enhanced contrast and, when applied after immunolabeling, did not appear to affect probe levels. Tannic acid also enhanced the staining of fixed tissue in LR Gold acrylic resin and LA-GMA, but not of glutaraldehyde-osmium-fixed tissue in epoxy resin, even after sodium metaperiodate treatment. Treatment of sections with tannic acid is recommended for contrast enhancement in immunogold studies when osmium and tannic acid post-fixation must be avoided and the tissue is embedded in methacrylate or acrylic resin.

Unfixed tissue for electron immunocytochemistry: a simple preparation method for colloidal gold localization of sensitive epitopes using ethanediol dehydration.

A quick, simple protocol is described for the preparation of tissue for electron immunocytochemistry without the use of fixatives or deleterious solvents. Fresh, normal human colon was rapidly dehydrated in ethanediol (ethylene glycol) then embedded directly in low-acid glycol methacrylate. Using both mono- and polyclonal antibodies, in conjunction with colloidal gold probes, a range of intra- and extracellular epitopes were localized; these epitopes included lysozyme, chromogranin, desmin and collagen IV. Overall, the tissue compared well with material fixed in glutaraldehyde, partially dehydrated and embedded in LR White acrylic resin. Ultrastructural detail was good and was further enhanced, without affecting probe density and epitope localization, by the addition of 1% tannic acid or 1% uranyl acetate to the dehydrant. The technique is applicable to a wide range of tissues, allowing excellent antigen retention which might prove useful for the immunolocalization of sensitive epitopes.

The use of inert dehydration and glycol methacrylate embedding for immunogold localization of glomerular basement membrane components.

A protocol is described for the preparation of human pathology specimens without fixation, in order to perform immunocytochemistry at an ultrastructural level. Using the technique in conjunction with immunogold labeling, the basal lamina components type IV collagen, laminin, and heparan sulfate have been demonstrated in glomerular capillary loops in stored frozen human renal tissue. Tissue was thawed and immediately dehydrated with the inert cryoprotectant ethanediol (inert dehydration) followed by embedding in low-acid glycol methacrylate polymerized using the accelerator n,n-dimethylaniline. Tissue processed in this way retained superior antigenic activity when compared with tissue reprocessed from wax blocks and embedded in low-acid glycol methacrylate. Inert dehydration is a technique useful for the localization of processing sensitive epitopes in routine fresh or frozen archival pathologic material. Furthermore, high probe densities can be achieved without recourse to etching or enzyme treatments.

Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques.

Immuno- and affinity probes are widely used in biology and medicine, and are becoming essential tools for the elucidation of cell structure and function. This article reviews and discusses the bewildering array of probes and preparation techniques now available for the investigation of sectioned material by transmission electron microscopy, with critical analysis of their merits. Emphasis is placed on immunogold probes and methods useful for routine preparation, gathering together information that may be used to improve labeling techniques. New data on inert dehydration for the localization of sensitive epitopes without chemical or cryofixation is presented.

Ultrastructural localization of lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution.

The distribution of lysozyme (muramidase) within eosinophil leukocytes situated in the lamina propria of human colon was studied by immunoelectron microscopy using a range of standard techniques. Tissue processed in a variety of glutaraldehyde- or paraformaldehyde-based fixatives was partially dehydrated and embedded in the acrylic resin LR White. Tissue thus treated showed lysozyme in pale cytoplasmic granules and the matrix of specific granules, but not in their crystalloids. Trypsinization of sections had little effect on this result, and tissues fixed in glutaraldehyde and embedded in Araldite showed a low level of reactivity with a similar distribution. After etching LR or Araldite sections with sodium metaperiodate, the pale granules and specific granule matrices became negative for lysozyme and the crystalloids became positive. Because crystalloids also were labeled with normal rabbit immunoglobulin after etching, this apparent redistribution of label could be due to nonspecific binding rather than exposure of masked epitope.

Giant cell fibroblastoma. A distinctive, recurrent tumor of childhood.

Giant cell fibroblastoma, which was first described by Shmookler and Enzinger in 1983, is a rare fibroblastic tumor occurring mainly in male patients younger than 10 years of age. Only 28 cases have been reported so far. This paper describes seven new cases which were referred in consultation between 1968 and 1985. Five of the seven patients were male; their ages at the time of first surgery ranged from 2 to 18 years, but six of the seven were younger than 4 years. Tumors were all superficial and were situated in the chest (2 tumors), neck, axilla, scrotum, thigh, and finger; they generally grew slowly, were poorly circumscribed, and measured from 1 to 5 cm in maximum dimensions. Grossly, they were described variously as gray, gelatinous, firm, white, fibrous, pink, and watery. Histologically, there were varying proportions of moderately cellular solid areas and angiectoid areas, both featuring distinctive fibroblastic cells with what appeared to be multiple nuclei arranged in florets or wreaths. By electron microscopy, each wreath proved to be an excessively convoluted, but single, multilobed nucleus. The nuclei of the tumor cells were slightly hyperchromatic and not bizarre or pleomorphic. In follow-up times of from 12 months to 11 years (median 31 months), only one tumor recurred locally and none metastasized. The comparatively low recurrence rate in this series may well increase if the patients are followed for a longer period. Giant cell fibroblastoma should probably be classified with other nonmetastasizing, locally recurring fibroblastic proliferations of youth and childhood such as juvenile aponeurotic fibroma and recurring digital fibrous tumor of infancy.

A perspective of chromosome displacement as a primary step in the induction of aneuploidy.

This paper considers whether the model proposing chromosome displacement as a primary step in the induction of mitotic aneuploidy (Ford and Roberts, 1983a) is consistent with experimental data relating to spindle structure, chromosomal attachment to spindles, the promotion of aneuploidy and the relative involvement of the different human chromosomes in aneuploidy. It is concluded that the model is consistent with all the parameters thus far tested for autosomal chromosomes.