Breast carcinoma. Correlations between visual diagnostic criteria for histologic grading and features of image analysis.

OBJECTIVE: To investigate the relevance of image analysis for grading breast carcinomas. STUDY DESIGN: The results of histologic grading were correlated with 18 features of image analysis, including SD. "Simple" characteristics, like area and perimeter, shape indices, optical density and textural features of nuclei from cancer cells, were analyzed. Hematoxylin-eosin-stained tissue sections of 67 cancer specimens were routinely used for the study. RESULTS: We found statistically significant correlations between overall histologic grading and the sum of its subscores and features of image analysis, especially nuclear area, nuclear perimeter and the diameter of the circumscribing circle (diametercirc), including their SDs. The visually and therefore subjectively assessed subscore of the nuclear pleomorphism of histologic grading significantly correlated with the features of image analysis, like nuclear area, nuclear perimeter, diametercirc, integrated optical density and correlation (and their SDs). There were significant relationships between the absolute numbers of mitoses per 10 high-power fields and nuclear area, nuclear perimeter and diametercirc (and their SDs). We did not observe a significant correlation between the subscore of tubule formation of histologic grading and any of the features of the image analysis studied. Furthermore, the correlations between the features of image analysis and the subscores of the visual histologic grading system were analyzed with respect to each other. The subscore of nuclear pleomorphism of histologic grading correlated best with overall grading (r = .72), whereas no significant correlation could be found between the subscores of nuclear pleomorphism and mitotic activity. CONCLUSION: Image analysis provides objectivity and reproducibility to the grading of breast carcinomas and thus could contribute to more individualized prognostication of the disease.

Characterization of bile ducts in primary biliary cirrhosis and other liver diseases by immunohistochemical demonstration of Lewis(a) or b antigen.

We investigated Lewis(a) and Lewis(b) expression of bile ducts in 68 specimens from various kinds of liver disease. In addition, the number of IgM and IgG synthesizing plasma cells in the hepatic inflammatory reactions were immunostained and counted. We found a statistically significant decrease in the number of bile ducts in PBC (primary biliary cirrhosis) in comparison with either chronic active or persistent hepatitis (CAH/CPH). Bile ducts could be detected easily and constantly by their Lewis antigen expression. Isolated bile duct epithelial cells not apparent in H&E sections could be identified by Lewis(a) and b immunostaining. The number of plasma cells in PBC was significantly different than in (CAH/CPH). A large number of IgM plasma cells was a characteristic feature of PBC. However, neither counting of Lewis(a) and b positive bile ducts nor counting of IgM plasma cells was of definite diagnostic significance in the individual clinical case, since no cut-off value could be determined above or below which a PBC was ruled out or proven.

[Interpretation of the estrogen receptor content of breast cancer]. / Zur Interpretation des Ostrogenrezeptorengehaltes in Mammakarzinomen.

The biochemical analysis of estrogen receptor (ER) content, using the DCC (dextran-coated charcoal) method, was compared with different plotting methods of the estrogen-receptor immunocytochemical assay (ER-ICA) in 80 primary breast cancers including 9 metastases under routine conditions. It was evident, that the determined content of estrogen receptors depends on the technique of measurement, as well as the microscopic organizations of the individual carcinomas and should be interpreted in respect of their content of stroma and if possible of tumour heterogeneity.

Application of the chromogenic reaction to conventional silver staining, the Ag-NOR staining and the silver-intensified immunogold technique.

The principle of the chromogenic reaction and the transformation of "black and white" histochemical staining results or immunohistochemical signals to coloured microscopic images is described. The chromogenic reaction was optimized and is, so far, possible with either cyan-blue or magenta-red reaction products. The application of the chromogenic reaction to conventional silver stain was optimal in the Lendrum staining resulting in red or blue stained reticulin fibres. The Ag-NOR staining of the nucleolus organizing region (NOR) could be transformed by the same reaction to coloured reaction products as well as the silver-intensified immunogold technique in immunocytochemistry.

Quantitative immunohistochemistry: standardization and possible application in research and surgical pathology.

We describe three different methods of calibration of quantitative immunohistochemistry, two of them based on self-made dye-agarose or antigen-agarose substrates. Quantification of immunostained objects (immunosignals) can be done by (1) microscope photometry (plug method) or scanning-photometry; (2) and densitometry of a television transformed microscope image. Both approaches are subjected to the different methods of calibration and three examples of application.

Immunocytochemical detection of sulphonylated DNA in tissue sections. An alternative to Feulgen staining of DNA.

We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2'-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.

Histological classification of synovitis.

A simple system of synovitis classification is proposed based on histomorphological evaluation of 400 random specimens of synovial tissue. It discriminates between two types of diagnosis: 1) one with a high degree of information, being called C-synovitis (synovitis with a characteristic or diagnostic histomorphological pattern), where the clinical diagnosis can be predicted on the basis of the histomorphological pattern, 2) a second type of diagnosis with a lower degree of information, being called NC-synovitis (synovitis with non-characteristic or non-diagnostic morphological findings in the synovial membrane). We subdivided NC-synovitis into 4 subgroups and determined the RA-probability of each subgroup in order to permit clinical use of the proposed histomorphological classification. In addition, by using immunoperoxidase staining methods for immunoglobulins the subgroups were shown to have different quantities of extravasally deposited immunoglobulins in the synovial membrane. The subgroup with the highest RA-probability also turned out to have the highest amount of extravasally deposited immunoglobulins.

[Clinical value of histomorphologic studies of joint capsules]. / Klinische Wertigkeit der histomorphologischen Untersuchungen von Gelenkkapseln.

The clinical significance of the histomorphological evaluation of synovial tissue from 389 patients with joint diseases was studied. From these data we conclude: (1) the histomorphological diagnosis in joint diseases is rarely of decisive value (6.2%); (2) in 23.1% of cases the histomorphological diagnosis is judged to be of clinical assistance; (3) the histomorphological grading of the inflammatory reaction in the joint under study corresponds in approximately two out of three cases to the clinical judgement.

Immunophotometric quantification of extravascular immunoglobulin deposits in the synovial membrane of patients with osteoarthritis and rheumatoid arthritis.

The amount of extravascular immunoglobulin deposits in the synovial membrane of patients with osteoarthritis and rheumatoid arthritis was studied in comparison with that of patients suffering from non-joint diseases. Immunoglobulin deposits were immunostained using the three-layer immunoperoxidase method. The staining results were quantified with the help of a microscope photometer connected with a scanning stage. Several experiments involving artificial test substrates, diseases with allegedly increased extravascular deposits or diseases not exhibiting extravascular deposits of immunoglobulins are used to validate the described microspectrophotometrical approach for measuring extravascular immunoglobulin deposits. The scanning photometry demonstrates significantly higher amount of extravascular immunoglobulin deposits in rheumatoid arthritis as compared with osteoarthritis and non-joint diseases.

Use of lectin-immunohistochemistry in joint diseases.

The lectin binding pattern of the inflamed synovial tissue of patients with OA (osteoarthritis) and RA (rheumatoid arthritis) were examined with 18 different lectins. The synovial lining cells are heavily stained by PHA-L, WGA, PNA, Con A, MPA and RCA. In the subsynovial layer, WGA, PHA-L and RCA were found to be fairly good monocytic markers. The lectin binding pattern of the lectins under study, however, failed to discriminate between patients with OA and RA.

Immunophotometric analysis of immunoglobulin synthesis and deposition in synovial membranes of patients with rheumatoid arthritis.

Immunophotometrical measurements of the Ig synthesis of plasma cells in joint capsules of patients with rheumatoid arthritis and osteoarthritis were compared. Patients with rheumatoid arthritis showed higher mean values in the single plasma cell than patients with osteoarthritis for all Ig isotypes except IgG. These results are indicative for a B cell activation with increased synthesis of Ig in RA.

In situ evaluation of immunoglobulin synthesis of synovial plasma cells in rheumatoid arthritis and osteoarthritis by plug photometry.

Synovial membranes of patients with osteoarthritis--a disease with unaltered immunoregulation--and rheumatoid arthritis were immunostained for the different immunoglobulin isotypes using the PAP-method and a two-layer alkaline phosphatase technique. Randomly selected plasma cells with positive immunostaining were measured by plug photometry. The average extinction of the single plasma cell is considered to be indicative of the immunoglobulin synthesis of the measured plasma cell and the isotype under study. When we compared both disease entities, patients with rheumatoid arthritis were characterized (i) by higher average extinction values for all Ig-isotypes except IgG (ii) by a higher rate of high producer plasma cells. From this data, we conclude that immunophotometry is a new in vivo method for discriminating two types of synovitis (i) one without increased Ig-synthesis at the single cell level and (ii) a second one with increased Ig-synthesis.

Colour development of immunogold-labelled antibodies for light microscopy.

We describe an improvement of the immunogold technique, which is based on the colour development of silver-intensified immunogold signals. This method (referred to as the coloured-SIG technique) was found to be as sensitive as the silver-intensified immunogold method and more sensitive (in two of the three tested systems) than immunoenzymatic procedures, such as the peroxidase/antiperoxidase technique or the avidin-biotin system. The coloured SIG-method results in either a magenta-red or a cyan-blue final reaction product. Therefore, this new improvement of the immunogold technique should be useful in double-staining methods, immunoblot methods and conventional immunostaining methods.

Distribution of albumin in normal and regenerating livers of mice. A light microscopic immunohistochemical and autoradiographic study.

The conditions affecting the immunohistochemical identification of albumin in livers of male NMRI-mice were investigated by light microscopy. In normal livers albumin is randomly distributed, revealing a pancytoplasmic nearly homogen reaction in groups of hepatocytes or single parenchymal cells. However, combined autoradiographic studies after pulse labelling with 3H-valine and perfusion experiments with human albumin indicate that this distribution is caused by albumin from blood plasma and does not reflect true protein synthesis. After perfusion of the livers followed by immunohistochemical amplification techniques which allowed to dilute the primary antibody up to 1:30,000, albumin could be detected nearly in all liver parenchymal cells as granular deposits decreasing in its density from periportal fields towards the terminal hepatic venules. In regenerating livers due to partial hepatectomy no remarkable differences in granular albumin deposits between G1- and S-phase of the cell cycle could be detected as was demonstrated by combined immunohistochemistry and 3H-dThd-autoradiography. However, during mitosis the content of albumin was often considerably reduced.

[Immunohistochemistry: theoretical potentials and practical application]. / Immunhistochemie: Theoretische Möglichkeiten, praktische Anwendung.

Immunhistochemical methods are increasingly used and their application in surgical pathology is obvious. Nine different immunohistochemical techniques are compared in this review article. The peroxidase anti-peroxidase method of Sternberger et al. (1970), the avidin biotin complex method of Hsu et al. (1981) and the labeled avidin biotin technique of Guesdon et al. (1979) are to be preferred. Two step methods using labeled antibodies such as alkaline phosphatase or peroxidase-labeled second antibodies are less sensitive. However, two-step methods using alkaline phosphatase-labeled antibodies offer the advantage of double staining procedures since alkaline phosphatase reacts with different azo dyes and results in a wide spectrum of colours in the final reaction product. At present, the major problems of immunohistochemistry in daily work are the selection of the fixative and of appropriate controls. Though both problems remain unresolved, immunohistochemical methods should be used for special problems in surgical pathology.

Distribution of mast cells in human synovial tissue of patients with osteoarthritis and rheumatoid arthritis.

Mast cells are demonstrated in synovial membranes of patients with osteo-arthritis and rheumatoid arthritis using a new staining principle based on interaction of heparin in mast cell granules with peroxidase labeled avidin. It was found that mast cell numbers in the subsynovial layer of patients with rheumatoid arthritis were significantly lower than those in patients suffering from osteo-arthritis. This decrease can be mainly attributed to patients with rheumatoid arthritis whose synovitis was characterized by a distinct histomorphological pattern consisting of lining cell ulcers and granulation tissue. However, when mast cell numbers in rheumatoid arthritis and osteo-arthritis patients were compared without respect to mast cell distribution in the subsynovial layer or the stratum fibrosum, no statistical differences between the diseases could be observed.