Ultrastructural postembedding immunogold labeling: applications to diagnostic pathology

Ultrastructural postembedding immunogold labeling combines the advantages of routine transmission electron microscopy and detection of antigenic epitopes. As a result, morphologic findings can be accurately interpreted. Localization of morphologic correlates for the various antigenic determinants allows a good definition of the applications and limits of immunocytochemistry in diagnostic surgical pathology. In some cases, immunofluorescence and immunocytochemistry results at light- microscopic levels can be either validated or determined to be nonspecific. Application of ultrastructural immunolabeling to surgical pathology is no longer a distant possibility but rather a reality. The technique provides reliable and reproducible results. Image analysis and microanalytical techniques applied to ultrastructural immunolabeling serve objectively to analyze and clearly to portray results. This paper describes experience with ultrastructural immunolabeling of various renal diseases and neoplasms by means of an easy-to-use methodology that can be applied to the evaluation of specimens in a diagnostic surgical pathology laboratory.     

Correlation of Subcellular and Intratumoral Photosensitizer Localization with Ultrastructural Features After Photodynamic Therapy

Photodynamic therapy (PDT) of cancer typically involves systemic administration of tumor-localizing photosensitizers followed 48-72 h later by exposure to light of appropriate wavelengths. Knowledge about the distribution of photosensitizers in tissues is still fragmentary. In particular, little is known as to the detailed localization patterns of photosensitizers in neoplastic and normal tissues as well as the relationship between such patterns and the actual targets for the photosensitizing effect. This review focuses on ultrastructural features seen in treated cells and tumors. An attempt is made to correlate these findings with the subcellular/intratumoral localization pattern of the photosensitizers in tumor cell lines in vitro and in tumor models in vivo. Several subcellular sites are main targets of PDT with different sulfonated aluminum phthalocyanines (AIPcS_n) in the human tumor cell line LOX. Nuclei are not among the primary targets. Overall, the ultrastructural changes correlate well with the data about the subcellular localization patterns for each analogue of AIPcS_n in the same cell line. Similar findings are also obtained for the family of sulfonated mesotetraphenylporphines (TPPS_n) in the NHIK 3025 cell line. The mechanisms involved in the killing of tumors by PDT seem to be a complex interplay between direct and indirect (via vascular damage) effects on neoplastic cells according to the intratumoral localization pattern of the applied dye. Several factors can affect the localization pattern of a drug, such as its chemical character, the mode of drug delivery, the time interval between drug administration and light exposure, and tumor type. Furthermore, whether local immune reactions (such as macrophages) and apoptosis (programmed cell death) are involved in the destruction of neoplastic cells by PDT in vivo is still an enigma. A general model for PDT-induced tumor destruction is suggested.     

An automated computer-assisted ultrasonic diagnostic system

All-Russian Scientific-Research Institute for Medical Instrument Engineering. Diagnostic Medical Systems Joint-Stock Company, Moscow. Translated from Meditsinskaya Tekhnika, No. 5, pp. 43–44, September–October, 1994.     

An automated cervical pre-cancerous diagnostic system

OBJECTIVE: This paper proposes to develop an automated diagnostic system for cervical pre-cancerous. METHODS AND DATA SAMPLES: The proposed automated diagnostic system consists of two parts; an automatic feature extraction and an intelligent diagnostic. In the automatic feature extraction, the system automatically extracts four cervical cells features (i.e. nucleus size, nucleus grey level, cytoplasm size and cytoplasm grey level). A new features extraction algorithm called region-growing-based features extraction (RGBFE) is proposed to extract the cervical cells features. The extracted features will then be fed as input data to the intelligent diagnostic part. A new artificial neural network (ANN) architecture called hierarchical hybrid multilayered perceptron (H(2)MLP) network is proposed to predict the cervical pre-cancerous stage into three classes, namely normal, low grade intra-epithelial squamous lesion (LSIL) and high grade intra-epithelial squamous lesion (HSIL). We empirically assess the capability of the proposed diagnostic system using 550 reported cases (211 normal cases, 143 LSIL cases and 196 HSIL cases). RESULTS: For evaluation of the automatic feature extraction performance, correlation test approach was used to determine the capability of the RGBFE algorithm as compared to manual extraction by cytotechnologist. The manual extraction of size was recorded in micrometer while the automatic extraction of size was recorded in number of pixels. Region color was recorded in mean of grey level value for both manual and automatic extraction. The results show that the estimated size and mean of grey level have strong linear relationship (correlation test more than 0.8) with those extracted manually by cytotechnologist. Hence, the size of nucleus, size of cytoplasm and grey level of cytoplasm created very strong linear relationship with correlation test more than 0.95 (approaching one). For the intelligent diagnostic, the performance of the H(2)MLP network was compared with three standard ANNs (i.e. multilayered perceptron (MLP), radial basis function (RBF) and hybrid multilayered perceptron (HMLP)). The performance was done based on accuracy, sensitivity, specificity, false negative and false positive. The H(2)MLP network performed the best diagnostic performance as compared to other ANNs. It was able to achieve 97.50% accuracy, 100% specificity and 96.67% sensitivity. The false negative and false positive were 1.33% and 3.00%, respectively. CONCLUSIONS: This project has successfully developed an automatic diagnostic system for cervical pre-cancerous. This study has also successfully proposed one image processing technique namely the RGBFE algorithm for automatic feature extraction process and a new ANN architecture namely the H(2)MLP network for better diagnostic performance.     

Ultrastructural immunolabeling in diagnostic surgical pathology: illustrative cases.

Ultrastructural immunolabeling techniques can be reproducibly applied to resolve diagnostic dilemmas in surgical pathology. The technique utilized for processing and labeling the cases presented in this article is simple and reproducible. The specimens were fixed in Carson-Millonig phosphate-buffered 4% formaldehyde fixative and embedded in LR White resin following a well-described protocol. Immunogold labeling allows precise localization of antigenic sites without compromise of the underlying ultrastructural morphologic findings; thus detailed immunomorphologic assessment is achieved. Immunoelectron microscopy techniques permit clarification of doubtful immunocytochemistry results by labeling antigenic epitopes in cases in which light microscopy immunocytochemistry techniques fail to show unequivocal positive results (probably as a consequence of small amounts of detectable antigen present in the tissues). Not only does ultrastructural labeling permit identification of focally positive reactions essential for accurate diagnosis in a given case, but it also localizes the antigenic determinants to specific cellular sites, providing immunomorphologic correlation. The specificity of the interpreted results can then be judged accordingly. Overall, ultrastructural immunolabeling is more sensitive than light microscopic immunocytochemistry. Ultrastructural immunolabeling can play a crucial role in the characterization of some tumors that cannot be accurately classified with other diagnostic techniques, even when combined.     

Dilated cardiomyopathy in infancy. Ultrastructural image analysis for diagnostic purpose

30 patients in paediatric age have been submitted to endocardial biopsy of the right ventricle, most of them affected by a dilated cardiomyopathy. Our aim was to improve knowledge on this disease using ultrastructural morphometric analysis. An enlargement of myocardial cells with granular aspects of the cytoplasm, known as cloudy swelling, was observed at histological level. Electronmicroscopy showed two main abnormalities: 1) a constant increase of the chondroma resulting in nuclear deformation and myofibrillary dislocation towards the sarcolemma. 2) disarrangement of thin (actin) and thick (myosin) myofilaments. Such myofibrillary dislocation, designated as fibrillar segregation, suggests how myocardial fibres may loose their ability to contract. The stereological study of these two ultrastructural abnormalities showed that mitochondria are increased in number and size and that the loss of contractile properties of myocardial cells can be related to myofibrillar segregation.     

Immunohistochemistry of nervous system antigens: diagnostic applications in surgical neuropathology

Immunohistochemical localization of nervous system antigens is becoming an increasingly useful tool in the diagnosis of neuropathologic lesions. Use of antigen markers specific for certain cell types, when properly employed, can confirm suspected diagnoses or solve difficult differential diagnoses. Presently the most useful markers are the glial antigens, glial fibrillary acid protein and S-100 protein, but a number of other markers, such as neurofilament proteins and neuron-specific enolase, have shown some diagnostic utility.     

Ultrastructural immunolabeling: a general overview of techniques and applications.

Ultrastructural immunolabeling techniques combine the advantages of routine electron microscopy and detection of antigenic epitopes, the latter of which is customarily done by immunocytochemistry at the light microscopic level. In surgical pathology, immunocytochemistry has become routine to approach the differential diagnosis of difficult cases. Immunoelectron microscopy has been used primarily for research purposes or for addressing specific questions in diagnostic pathology. Ultrastructural immunolabeling is extremely useful in those instances in which the ultrastructural and immunocytochemical findings are not pathognomonic and are subject to interpretation. Preembedding and postembedding labeling techniques have been described in the literature. Preembedding techniques are not as applicable to diagnostic work, however. Their use remains rather limited to applications in which the antigen to be labeled cannot maintain its viability when exposed to fixatives. Even in such cases a new methodology--the LifeCell process--has emerged; this technique cryofixes tissues, thereby maintaining antigenic integrity. After cryofixation, a postembedding labeling technique can be utilized. Immunogold and peroxidase methods are used for labeling. Immunogold methods elegantly mark reaction sites with preservation of underlying morphology. Postembedding immunogold methods are used by most individuals working in the field. Ultrastructural labeling techniques are rapidly moving from classification as exclusively research tools to the diagnostic arena.     

A starch-based microparticulate system dedicated to diagnostic and therapeutic nuclear medicine applications

The aim of this work was to develop a new microparticulate system able to form a complex with radionuclides with a high yield of purity for diagnostic or therapeutic applications. Owing to its properties potato starch was chosen as starting material and modified by oxidization and coupling of a ligand (polyamine) enabling modified starch to chelate radionuclides. The choice of suitable experiments was based on a combination of a Rechtschaffner experimental design and a surface response design to determine the influence of experimental parameters and to optimize the final product. Starch-based microparticle formulations from the experimental plans were compared and characterized through particle size analysis, scanning electron microscopy, elemental analysis and, for the most promising formulations, by in?vitro labeling stability studies and determination of free polyamine content or in?vivo imaging studies. The mechanism of starch-based microparticle degradation was identified by means of size measurements. The results of the Rechtschaffner design showed the positive qualitative effect of the temperature and the duration of coupling reaction whereas surface response analysis clearly showed that, by increasing the oxidization level and starch concentration, the nitrogen content in the final product is increased. In vitro and in?vivo characterization led to identification of the best formulation. With a size around 30?μm, high radiochemical purity (over 95%) and a high signal-to-noise ratio (over 600), the new starch-based microparticulate system could be prepared as ready-to-use kits and sterilized without modification of its characteristics, and thus meet the requirement for in?vivo diagnostic and therapeutic applications.     

Clear Cell Sarcoma. An Immunohistochemical and Ultrastructural Study

The histogenesis of clear cell sarcoma was investigated by immunohistochemical examination of five tumors (two melanotic and three amelanotic) and electron microscopic examination of two of these tumors (one melanotic and one amelanotic). Melanin production was observed histologically in two of the tumors. The cytoplasm of ceils in both types of tumor contained various numbers of melanosomes. Melanoma-specific antibody (HMB-45), anti S 100 protein, and anti vimentin antibodies gave positive reactions in four tumors, while all tumors showed Leu 7 immunoreactivity. No cytokeratin or epithelial membrane antigen (EMA) was detected immunohisto-chemically in any tumor. The immunoreactivity of this type of tumor with HMB-45 antibody strongly suggests melanocytic differentiation rather than schwannian or synovial differentiation. The reaction of the cells of one tumor with only Leu-7 indicates the existence of undifferentiated clear cell sarcoma of neuroectodermal origin that does not show definite melanocytic differentiation. Acta Pathol Jpn 39: 321 327, 1989.     

HUNTER'S SYNDROME. An Ultrastructural Study of an Autopsy Case

An autopsy case of a 10-year, 8-month-old boy with Hunter's syndrome Is reported with emphasis on the ultrastructural findings of almost all the organs, except the brain. Intracytoplasmic inclusion bodies were observed in various organs as follows: nerve cells and glia in the spinal cord, hepatocytes and Kupffer cells in the liver, sinusoidal endothelium of the spleen, proximal tubules, podocytes and epithelium of Bowman's capsule of the kidney, interstitial fibroblast-like cells among cardiac muscle bundles, cardiac valves and aorta, exocrine and endocrine cells of the pancreas, adrenocortical cells, follicular epithelial cells of the thyroid, Leydig cells of the testis, chondrocytes, fibroblasts and endothelium of capillaries throughout the body. Three types of inclusion bodies were morphologically distinguishable. Type 1: clear vacuole, Type 2: zebra body, Type 3 : clear vacuole with a lipid-like lamellar structure. The clear vacuole (Type 1) was thought to represent an accumulation of glycosaminoglycans, and the zebra body (Type 2), probably ganglioside. The type 3 inclusion body might be an intermediate and mixed form of the type 1 and type 2 inclusions. Histochemical study also suggested that the type 3 inclusion body contained glycosaminoglycan and a type of lipid. ACTA PATHOL JPN 38: 1175 ∼ 1190, 1988.     

Ultrastructural alterations in livers with metastasis.

The electron microscopic examination of 13 biopsy specimens of livers that contained metastatic carcinoma revealed degenerative alterations of the mitochondria, cystic dilatation of the endoplasmic reticulum, increased amounts of osmiophilic material, some in various stages of autophagocytosis, and lipid vacuoles within the hepatocytes. In ten of 13 cases, the hepatocytes demonstrated mitochondrial paracrystalline inclusions. The observation of the latter structures in edematous hepatocytes containing phospholipid-rich osmiophilic deposits tends to confirm the experimental hypothesis that paracrystalline inclusions are caused by the precipitation of unmasked phospholipids. The spectrum of ultrastructural findings suggests hypoxia and impaired lipid metabolism of the hepatic tissue as a result of congestion and bile stasis caused by the tumor nodules.