Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections.

AIMS: To evaluate the performance of an immunoperoxidase stain using the monoclonal antibody EB-A1 to detect Aspergillus species in formalin fixed, paraffin wax embedded tissue. METHODS: The monoclonal antibody EB-A1 directed against galactomannan was used to detect Aspergillus species in 23 patients with suspected or confirmed invasive aspergillosis. Immunostaining was performed on formalin fixed, paraffin wax embedded tissue using the streptavidin-biotin method and compared with conventional haematoxylin and eosin, periodic acid-Schiff, and Gomori-Grocott stains. Results of immunostaining were semiquantitatively analysed with regard to both intensity of staining and number of positively staining micro-organisms. Tissue sections from 16 patients with confirmed invasive mycoses due to Candida species, Apophysomyces elegans, Rhizopus oryzae, Pseudallescheria boydii and Histoplasma capsulatum were used as controls. RESULTS: In 19 (83%) of 23 cases invasive aspergillosis was confirmed by both histological examination and culture (18 Aspergillus fumigatus and one A flavus). Immunoperoxidase stains were positive in 17 (89%) of 19 cases including one case of disseminated infection due to A flavus. Furthermore, the immunoperoxidase stain was positive in a culture negative tissue section with histological evidence of mycelial development, indicating the presence of Aspergillus species. Some cross-reactivity was observed with the highly related fungus P boydii, although the number of mycelial elements that stained was low. CONCLUSIONS: Immunoperoxidase staining using the monoclonal antibody EB-A1 performs well on routinely processed tissue sections and permits detection and generic identification of Aspergillus species, although it was no better than conventional histopathology in identifying the presence of an infection. An additional advantage is that the immunostain may help to provide an aetiological diagnosis when cultures remain negative.     

Immunocytochemical method for the detection of terminal deoxynucleotidyl transferase in acute leukemia

Summary Terminal deoxynucleotidyl transferase (TdT) has become an important marker for the classification of acute leukemias. Originally detected by a biochemical assay, the presence of this enzyme in leukemia can now be detected by making use of a highly specific antibody and by applying the immunofluorescence method. In this study, an attempt was made to apply the unlabeled peroxidase-antiperoxidase (PAP) technique for the examination of TdT positive cells. We have optimized this method and have compared the different assays — biochemical, immunofluorescence, and immunocytochemical (PAP) — in ten patients with acute leukemia. The results obtained from all three methods are in accordance with one another, but the PAP method is a further simplification for the detection of TdT in the routine diagnostic of acute leukemias.     

An enhanced immunocytochemical method for staining bone marrow trephine sections.

AIMS: The detection of cellular antigens in fixed decalcified bone marrow trephine (BMT) sections depends on the method of processing, the nature of the antigen and antibody, antigen retrieval techniques, and the sensitivity of the immunocytochemical method. This study evaluated a tyramide enhanced avidin-biotin immunostaining method on formalin fixed decalcified BMT sections to determine whether the method could detect previously undetectable antigens. METHODS: Nineteen BMT biopsies from a range of haematological disorders were evaluated with 43 antibodies to haemopoietic antigens using horseradish peroxidase and alkaline phosphatase detection methods, using the tyramide enhanced avidin-biotin immunostaining method. RESULTS: Compared with standard avidin-biotin immunostaining methods the tyramide enhanced immunostaining method showed enhanced signal intensity, gave positive labelling for antigens that require pretreatment by other methods, and previously unreactive antigens were detected. Primary antibodies could be used at up to 200 times higher dilutions. CONCLUSION: The tyramide enhanced immunostaining method, while retaining specificity, is highly sensitive and enables an increased number and range of antigens to be detected than previously possible. The method could be applied to BMT sections for the routine diagnosis and classification of haematological disorders.     

A method to generate antigen-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections

Abnormalities in HLA class I antigen expression are frequently found in malignant tumors. Their potential role in the clinical course of the disease and in the outcome of T cell-based immunotherapy has stimulated interest in the characterization of the molecular mechanisms underlying HLA class I antigen abnormalities in malignant cells. Multiple mechanisms have been identified. Among them are abnormalities in antigen processing machinery (APM) component expression. In spite of this information, APM component expression in malignant lesions has been investigated only to a limited extent because of the lack of availability, for most APM components, of monoclonal antibodies (mAb) which stain formalin-fixed, paraffin-embedded tissues. The latter are the substrate of choice in immunohistochemical (IHC) reactions. To overcome this limitation, we have developed a simple and reproducible method to generate APM component-specific mAb which stain formalin-fixed, paraffin-embedded tissue sections. This method involves five steps: (i) immunogenic amino acid sequences, which display low homology with their mouse counterparts when possible, are identified in APM components and utilized to synthesize peptides; (ii) BALB/c mice are immunized with keyhole limpet hemocyanin (KLH)-conjugated synthetic peptides and with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-purified recombinant APM component proteins; (iii) immunized mice, which develop high titer APM component-specific antibodies, are utilized to generate hybridomas which are screened for APM component-specific antibody production by Western blotting assays, with lymphoid cell lysates; (iv) identified APM component-specific mAb are characterized in their specificity and in their reactivity with permeabilized cells in ELISA and/or flow cytometry; and (v) mAb, with the appropriate reactivity pattern, are tested in IHC reactions with formalin-fixed, paraffin-embedded tissue sections. The use of the methodology we have developed resulted in the generation of a panel of APM component-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections in IHC reactions. These reagents will facilitate the analysis of APM component expression in tissues under physiological and pathological conditions. In addition, the methodology we have described is likely to be applicable to other antigenic systems to develop mAb capable of detecting protein components of interest in formalin-fixed, paraffin-embedded tissue sections.     

Immunohistochemical demonstration of mitochondria in routinely processed tissue using a monoclonal antibody

We report results obtained using the monoclonal antibody M-II 68, which recognizes inner mitochondrial membrane in routinely processed (formalin-fixed and paraffin-embedded) tissue by light microscopic immunohistochemistry. In ten normal brains, the range of immunoreactivity in various cell types and locations was defined. The most intense staining was observed in Purkinje cells, in neurons of cranial nerve nuclei, pons and substantia nigra, as well as in choroid plexus epithelial cells. By comparison with this control group, one case of primary mitochondrial encephalomyopathy exhibited increased staining of endothelial and vascular smooth muscle cells, choroid plexus epithelial cells, and neurons of various locations. Scattered ragged-red fibres were heavily labelled in one case of mitochondrial myopathy, while ten muscles without mitochondriopathy were left unstained. Our method is able to detect accumulations of mitochondria and increases in mitochondrial cristae density. It could prove useful for differential diagnosis of routine biopsy material and for clarification of cell types involved in mitochondrial cytopathies.     

Detection of messenger RNA in routinely processed tissue sections with biotinylated oligonucleotide probes

In situ hybridization (ISH) with a radioactively 35S-labeled probe and a biotinylated oligonucleotide probe for human calcitonin was used to analyze eight cases of medullary thyroid carcinoma (MTC) in paraffin sections. Three of these cases were also studied with frozen tissue sections. The biotinylated probe readily detected calcitonin messenger RNA (mRNA) in routinely processed formalin-fixed paraffin-embedded tissue section within 24 hours. Northern hybridization and other control studies demonstrated the specificity of the calcitonin probe. Biotinylated oligonucleotide probes for other mRNAs present in high abundance such as adrenocorticotroipic hormone (ACTH), prolactin, and growth hormone also detected the respective mRNAs in pituitary tissues. These result show that biotinylated oligonucleotide cDNA probes can be used to detect specific mRNAs present in large amounts in some endocrine cells and tumors by ISH. This approach offers an alternative that does not require the use of molecular cloning or radioactive probes for this investigative and diagnostic technique.     

Ki-M6 immunostaining in routinely processed sections of reactive and neoplastic human lymphoid tissue

A monoclonal antibody, termed Ki-M6 (CD68), which shows a restricted reactivity to cells of the monocyte/macrophage system, has been evaluated primarily with the use of cryostat sections. In this study the authors could assess that the Ki-M6 antibody recognizes a fixation-resistant epitope in most human macrophages. The Ki-M6 immunoreactivity with monocyte/macrophage-related cells was established by testing on routinely processed samples of reactive and neoplastic lymphoid tissues; it was compared with the staining for vimentin (V9) and S-100 protein antibodies, with visualization of the stationary elements of lymphoid tissues, with the aim of establishing its value in the study of the nonlymphoid microenvironment. The Ki-M6 antibody reactivity could be achieved with Bouin-fixed, paraffin-embedded tissue sections, without any proteolytic treatment, with the use of the avidin-biotin complex (ABC) method, especially after overnight incubation time at 4 degrees C. Some reduction in antigenic reactivity was observed in B5- or formaldehyde-fixed samples. The antibody reacted with macrophages of all different lymph node compartments; a broad reactivity against cells of macrophage lineage, including multinucleated giant cells, was observed in epithelioid granulomas. Ki-M6-positive cells other than classic macrophages were the so-called "plasmacytoid T-cells" and cells displaying elongated cytoplasms with fibroblastic-like features. Granulocytes, follicular dendritic reticulum cells, and interdigitating reticulum cells did not reveal any reactivity with Ki-M6 antibody. In malignant lesions, neoplastic cells of follicular and diffuse B- and T-cell lymphomas, including large cell non-Hodgkin's lymphomas, and Reed-Sternberg cells of Hodgkin's disease were negative in all cases studied. This study shows that Ki-M6 seems to be another anti-macrophage-specific antibody that reacts, in routinely processed tissue sections, with tissue macrophages but not with accessory cells. Thus, it may be a valuable addition to vimentin and S-100 protein antibodies for investigation of the microenvironmental organization of lymphoid tissues both in normal and neoplastic conditions.     

Simultaneous evaluation of terminal deoxynucleotidyl transferase and myeloperoxidase in acute leukemias using an immunocytochemical method

The classification of acute leukemia is important for the selection of optimal therapy. Classification often rests on morphologic, cytochemical, and immunologic criteria, and the marker enzyme terminal deoxynucleotidyl transferase (TdT) has been considered to be a reliable indicator of lymphoblastic leukemias. Because TdT-positive cells sometimes are seen in leukemias otherwise identified as myeloblastic, the authors evaluated blasts identified as myeloid by the presence of myeloperoxidase (MPO) for the simultaneous expression of TdT. The blasts in the bone marrow aspirate or peripheral blood of unselected patients with hematologic malignancies were evaluated and 60 cases are shown. The French-American-British system and, in some patients, cytochemical and immunologic studies were used to classify the leukemias. The authors demonstrated that blasts simultaneously contained MPO and TdT in 29% of patients with acute myeloblastic leukemia and 3% of patients with acute lymphocytic leukemia (ALL). This finding supports the hypothesis that TdT is an expression of cell primitivity rather than a marker for lymphoblastic cells.     

Modified hematoxylin and eosin staining method for epoxy-embedded tissue sections

A modified hematoxylin and eosin staining method for glutaraldehyde fixed, osmium tetroxide postfixed, and epoxy resin (Medcast Resin) embedded tissue is described. Two microns thick sections of human and animal tissues are treated with 4% H2O2 for 4 minutes, washed, dryed, and then flooded with Gill's III hematoxylin in a moist chamber at 37 degrees C for 90 minutes. Subsequent steps are: washing; bluing in ammonia water for 1 minutes; washing again and staining in 1% eosin Y alcoholic solution for 5 minutes; rinsing in 100% ethanol; drying and mounting with Permont. This procedure is quick, easy and successful in demonstrating both color scale and quality similar to hematoxylin and eosin stains obtained in standard paraffin sections.     

An improved method for the species-specific assessment of mycobacteria in routinely formalin-fixed and paraffin-embedded tissues

A polymerase chain reaction (PCR) assay for the rapid and species-specific diagnosis of mycobacterial infections in paraffin-embedded clinical specimens was developed using oligonucleotide primers to amplify a fragment of the DNA coding for the ribosomal 16S RNA of mycobacteria. The oligonucleotide primers amplified DNA from all 14 species of mycobacteria tested. By means of a reamplification protocol, as few as one to two mycobacteria could be detected in the presence of human DNA. The method of DNA isolation and amplification was applied on sections of routinely formalin-fixed and paraffin-embedded tissues. PCR for the β-actin gene served as a control for successful DNA isolation. Mycobacterial DNA could be detected in cases of mycobacterial infections. The mycobacterial species was determined by additional sequencing of the PCR fragment. This PCR method may be a powerful tool for the diagnosis of mycobacterial infections from histopathological material and for the assessment of those mycobacteria that cannot readily be cultured, such as Mycobacterium leprae.     

Method for the simultaneous labelling of terminal deoxynucleotidyl transferase (TdT) and membrane antigens.

A method for the simultaneous labelling of terminal deoxynucleotidyl transferase (TdT) and membrane antigens is described. TdT is visualised in the cell nucleus with the peroxidase-antiperoxidase (PAP) method, and the immunogold method is used in combination with monoclonal antibodies against membrane antigens. The morphology of the labelled cells is well preserved for analysis by light microscopy and the preparations obtained can be kept permanently. This method is useful for the analysis of mixed cell proliferations, particularly in leukaemias.     

Monoclonal antibodies reactive in routinely processed tissue sections of malignant lymphoma, with emphasis on T-cell lymphomas

The immunoreactivity of eight monoclonal antibodies was evaluated on 45 routinely processed lymphomas (22 T-cell lymphomas, 11 B-cell lymphomas, and 12 cases of Hodgkin's disease). Two antibodies reactive with leukocyte common (T200) antigens (PD7/26 and 2B11) stained most of the B- and T-cell lymphomas but did not stain the Reed-Sternberg cells and variants in Hodgkin's disease. Two antibodies known to stain B cells (LN-1 and LN-2) reacted with some of the B-cell lymphomas, but LN-2 also reacted with the neoplastic cells in six of 22 T-cell lymphomas and with the Reed-Sternberg variants in eight of 12 cases of Hodgkin's disease. The granulocyte antibody anti-Leu M1 reacted with most cases of Hodgkin's disease but also reacted with two of 11 B-cell non-Hodgkin's lymphomas. An antibody to epithelial membrane antigen (anti-EMA) stained some cases of T-cell lymphoma, B-cell lymphoma, and Hodgkin's disease. Leu 7 was expressed in one T-cell lymphoma and in one case of Hodgkin's disease. A novel antibody reactive with T cells (L60) stained all cases of T-cell lymphoma but also stained some cases of B-cell lymphoma and one case of Hodgkin's disease. We conclude that none of these antibodies, when used alone on routinely fixed paraffin-embedded material, is completely sensitive and specific for T-cell lymphoma, B-cell lymphoma, or Hodgkin's disease. However, a panel of antibodies is useful in distinguishing Hodgkin's disease from non-Hodgkin's lymphoma and in suggesting the B- or T-cell phenotype of non-Hodgkin's lymphomas.     

Avidin-biotin amplification procedures for the detection of human terminal deoxynucleotidyl transferase in cell smears

The authors have developed fluorescent avidin and avidin-peroxidase assays to detect human terminal deoxynucleotidyl transferase (TdT) in cell smears. These assays used specifically purified rabbit anti-calf TdT antibody, biotinylated goat anti-rabbit IgG antibody, and either fluorescein isothiocyanate-avidin or avidin-biotinylated horseradish peroxidase complex. The use of phosphate-buffered saline rather than TRIS-buffered saline or borate-buffered saline was essential to obtain optimal results in the fluorescent avidin assay. Fixation of cells with either 0.5% paraformaldehyde or 10% neutral buffered formalin was found to be superior to either no fixation or fixation with cold methanol or cold 0.1% glutaraldehyde in ethanol. The sensitivities of the fluorescent avidin and avidin-peroxidase assays were shown to be identical, based on staining intensities and percentages of positive cells when both assays were performed on cells from 14 patients with acute lymphoblastic leukemia (ALL), and 5 patients with lymphoblastic lymphoma (LL).