Cytomegalovirus detection by nonisotopic in situ DNA hybridization and viral antigen immunostaining using a two-color technique

Rapid methods of specific viral diagnosis in formalin fixed, paraffin embedded tissues include identification of viral incusions in routinely stained histologic sections, immunologic staining of viral antigens, and in situ nucleic acid hybridization. To correlate in situ hybridization with immunologic detection methods, sequential two-color staining was used on tissues from 12 patients, each containing characteristic cytomegalovirus (CMV) inclusions, using a biotinylated CMV DNA probe in an avidin-alkaline phosphatase-linked reaction followed by avidin-biotin complex immunoperoxidase staining of CMV antigen. CMV genetic material was seen in all 17 tissues. CMV antigen was detected in 11 of 17 tissues (65%). The DNA hybridization technique provided more intense staining, detected greater numbers of inclusions, and had less background staining than the immunoperoxidase technique. The alkaline phosphatase reaction product was stable through subsequent immunostaining steps, and immunologic reactivity of CMV antigen was not significantly reduced by prior hybridization steps. CMV DNA probe was localized predominantly within cell nuclei, while CMV antigen immunostaining was predominantly cytoplasmic. It was concluded that sequential in situ hybridization and immunocytochemistry can be performed on standard histologic sections. Furthermore, it is likely that the majority of CMV nucleic acid detected by this tissue hybridization technique is unencapsidated, intranuclear viral DNA and not DNA contained within complete CMV nucleocapsids.     

Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy

AIM: At present, the diagnosis of muscular dystrophy is made by means of immunohistochemistry on frozen sections. The aim of this study was to develop a sensitive and reproducible immunohistochemical method for use on formalin fixed, paraffin wax embedded sections for the demonstration of dystrophin associated proteins and other muscle associated antigens. METHODS: All the cases studied were from the files of the department of histopathology, Great Ormond Street Hospital for Children NHS Trust. Immunohistochemistry was performed on paraffin wax embedded sections with heat mediated antigen retrieval and overnight incubation with the antibodies at room temperature. Four different pretreatment buffers were tested in the attempt to optimise the immunostaining. Frozen sections were run in parallel for direct comparison. RESULTS: All the antibodies except delta sarcoglycan gave strong, consistent immunostaining in paraffin wax embedded sections, comparable with the frozen sections. The most consistent results were obtained using citrate/EDTA as the pretreatment buffer. CONCLUSION: A reliable and reproducible technique has been established, using a heat mediated citrate/EDTA buffer antigen retrieval method, which works well for most of the antibodies needed to make the diagnosis of muscular dystrophy in formalin fixed, paraffin wax embedded sections. This technique overcomes some of the inherent problems encountered using frozen muscle tissue and it could become a valuable tool for the diagnosis of muscular dystrophy.     

Immunohistochemical localization of human and simian immunodeficiency viral antigens in fixed tissue sections.

Antigens of human (HIV) or simian immunodeficiency viruses (SIV) were identified with polyclonal or monoclonal antibodies and avidin-biotin complex (ABC) immunohistochemistry in fixed surgical pathology and autopsy specimens of humans or monkeys with the acquired immunodeficiency syndrome. With B-5 fixative, viral antigens were readily detected in lymph nodes of 8 of 13 patients with follicular hyperplasia, but in only 1 of 12 patients with follicular atrophy. Antigen was detected in follicular dendritic reticular cells and rare blastlike cells, extracellularly, and in postcapillary venules, medullary lymphocytes, sinus histiocytes, and macrophages in some lymph nodes. In the brain at autopsy, antigen could be found in gliomesenchymal-cell nodules, astrocytes, vascular endothelial cells, multinucleated cells, and astrocytes and macrophages associated with demyelination. In contrast, 4 rhesus monkeys with experimental SIV infection had abundant antigen in sinus histiocytes, macrophages, and multinucleated giant cells of lymph nodes and spleen and in thymic epithelial cells. Brain lesions of monkeys resembled those of humans, with antigen found in macrophages and multinucleated giant cells. Antibodies to HIV also were immunoreactive in formalin-fixed tissue sections of monkeys containing SIV antigens. The ABC technique provided a fast and efficient method for localizing HIV and SIV antigens in fixed surgical and autopsy specimens. These findings are consistent with those found with in situ hybridization, ultrastructural studies, frozen sections of lymph nodes, and permanent sections of brain.     

Use of APAAP technique on paraffin wax embedded bone marrow trephines.

The immunoalkaline phosphatase (APAAP) technique was applied to the labelling of decalcified sections of formalin fixed, paraffin wax embedded bone marrow trephine biopsy specimens. A panel of monoclonal antibodies reactive with haemopoietic and epithelial antigens, which survive routine formalin fixation, was assessed on 72 cases of haematological malignancy (including acute and chronic leukaemias and lymphomas) showing bone marrow infiltration. The APAAP method showed clear distinct labelling of antigen positive cells without loss of antigens due to decalcification. Both normal or reactive single cells present in the sample and neoplastic cell populations could be identified morphologically and their antigenic phenotype and cellular origin, whether lymphoid or myeloid, established. The application of the APAAP method to routinely prepared paraffin wax embedded trephines has many advantages over the assessment of specially prepared cryostat sections of bone marrow.     

Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol

Specimens of bone marrow trephine biopsy (BMT) are transported and fixed in acetic acid-zinc-formalin fixative, decalcified in 10% formic acid-5% formaldehyde and processed with other specimens to paraffin-wax embedding. Sections, 1-microm-thick, are cut by experienced histotechnologists and used for haematoxylin and eosin, Giemsa, reticulin silver and other histological stains. Further, all immunohistochemical procedures used in the laboratory, including double immunostaining, can be used on these sections with no or minimal modifications. About 10,000 BMT specimens have been analysed using this procedure since 1997 and diseases involving the bone marrow have been classified successfully. More recently, standardised polymerase chain reaction-based analysis and mRNA in situ hybridisation studies have been conducted. Excellent morphology with good antigen, DNA and RNA preservation is offered by the Hammersmith Protocol.     

Comparison of peroxidase-antiperoxidase and avidin-biotin complex methods for the detection of papillomavirus in histological sections of the cervix uteri

A study was undertaken to determine the relative sensitivities of the peroxidase-antiperoxidase (PAP) and avidin-biotin complex (ABC) methods for the detection of human papillomavirus (HPV) antigens in acetic acid-ethanol fixed paraffin-embedded cervical tissue. Tissue sections prepared from 14 women suspected to have HPV infections with either atypia or dysplasia were stained immunohistochemically using an antiserum against genus-specific (common) antigen of bovine papillomavirus. Detection of HPV antigen was approximately twice as frequent by the ABC method as by the PAP method. Of the 14 cases studied, 43% were found to be HPV positive by the PAP method whereas 79% were HPV positive by the ABC method. In addition, the number of cells found to be HPV positive by the ABC method was approximately double the number by the PAP method.     

Surface membrane staining of immunoglobulins in paraffin sections of non-Hodgkin's lymphomas using immunogold-silver staining technique.

The immunogold-silver staining (IGSS) method is a new immunostaining technique with much enhanced sensitivity for demonstration of antigens in paraffin sections. A series of 10 non-Hodgkin's lymphomas of B cell type were stained for surface membrane immunoglobulins by the IGSS and peroxidase-antiperoxidase (PAP) methods using paraffin sections and polyclonal primary antisera. The resulting staining patterns were compared with those obtained using frozen sections of the same tissues, monoclonal antibodies and the immunoperoxidase technique. The IGSS method gave a clear demonstration of surface membrane immunoglobulins in neoplastic lymphocytes using paraffin sections and the pattern of staining achieved was comparable to that obtained by the immunoperoxidase technique employing frozen sections and monoclonal antibodies. PAP staining of paraffin sections consistently failed to demonstrate the presence of any surface membrane immunoglobulin. The IGSS method provides a new approach to the diagnosis of B cell lymphomas in which routinely fixed and processed tissues may be employed to demonstrate monoclonality.     

Immunohistochemical analysis of decalcified paraffin-embedded human bone marrow biopsies with emphasis on MHC class I and CD34 expression

 

Aims:

To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods.  

Methods and results:

Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and α-smooth muscle actin (α-SMA) required no pre-treatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1.  

Conclusions:

This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1–1.5 h.     

Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders.     

An evaluation of the immunohistochemistry benefits of boric acid antigen retrieval on rat decalcified joint tissues

This paper describes the evaluation and optimisation of boric acid antigen retrieval (AR) in rat joint tissue immunohistochemistry (IHC), with reference to two sample IHC targets, CD31 (PECAM-1) and Proliferating Cell Nuclear Antigen (PCNA). Sections of buffered formalin-fixed arthritic tibial/talus joints, decalcified with EDTA, EDTA/formalin or Surgipath(R) Decalcifier I(R), were subjected to one of a number of pre-treatments (none, 0.1% trypsin, 0.2 M acetic acid pH 7.0 or 0.2 M boric acid pH 7.0) and then immunostained for CD31 or PCNA. Of the pre-treatment AR regimens, boric acid gave the most consistent and specific immunostaining of both antigens in joints from the three different decalcification protocols. Satisfactory CD31 and PCNA staining was also achieved in EDTA decalcified joints with no pre-treatment. Likewise, PCNA could be demonstrated in Surgipath(R) decalcified tissue without pre-treatment, albeit at slightly lower staining intensity than achieved following boric acid. The remaining decalcification/pre-treatment conditions were unsatisfactory for IHC of the two antigens investigated because of lack of staining, non-specific staining or consistent loss of sections from the slides. Boric acid pre-treatment provides a valuable alternative low temperature AR method where conventional heat-mediated AR methods are normally required but cannot be used due to tissue type.     

Microwave antigen retrieval for immunocytochemistry on formalin-fixed,paraffin-embedded post-mortem CNS tissue

Microwave antigen retrieval methods were assessed for a panel of antibodies on formalin-fixed, paraffin-embedded sections from a range of human central nervous system (CNS) tissues taken at post-mortem. The immunoreactivity of a number of antigens (synaptophysin, PGP9.5, GFAP, carbonic anhydrase II, CD68, ferritin, HLA-DR, αβ-crystallin, mealses and herpes viruses) was markedly enhanced by this procedure compared with other methods of antigen unmasking, such as trypsinization. Enhancement was noted both by an increased number of positive cells and by the intensity of reactions within individual cells and their processes. Furthermore, the microwave method produced uniform immunostaining over large surface area sections with no loss of morphological detail. Large cryostat sections of CNS tissue can be difficult to obtain with good morphology. The ability to retrieve a wide range of antigen in formalin-fixed, paraffin-embedded CNS tissue will greatly increase the range of investigations that can be carried out on this type of stored material.     

Detailed phenotypic analysis of B-cell lymphoma using a panel of antibodies reactive in routinely fixed wax-embedded tissue.

Sixty-three well characterized B-cell lymphomas, with diagnoses previously established by conventional cryostat immunocytochemistry or limited paraffin immunocytochemistry, were studied. The tumors encompassed most of the Kiel subtypes and included the newly recognized entity, sclerosing mediastinal B-cell lymphoma. by the avidin-biotin peroxidase complex technique, each tumor was stained with a panel of monoclonal and polyclonal antibodies reactive in routinely fixed wax-embedded tissues. The panel included four reagents recognizing probable T-cell and B-cell restricted leukocyte common moieties (UCHL1, MT1, MB1, 4KB5), three antibodies to B-cell-related antigens (KiB3, MB2, LN1), and one to a macrophage-related antigen (Mac411). Other antibodies employed included anti-mu chain, anti-kappa, anti-lambda, and seven antibodies to non-phenotype-associated antigens, including HLA-DR (TAL-1B5, LN3, LN2, MB3), CD15 (C3D-1), and CD30 (BER-H2). Monotypic surface or perinuclear space and cytoplasmic immunoglobulin were detected in 80% of cases. Distinctive immunocytochemical profiles were demonstrable in many tumor categories by means of the panel of antibodies, thus facilitating the differential diagnosis of tumors of similar morphology. These results, together with our work on T-cell lymphoma in paraffin sections, show that accurate phenotypic analysis of lymphoma is now possible in routinely processed tissues.     

SSPE in Slovakia: immunocytochemical study.

In a retrospective study of two patients from Slovakia with clinical, virological and histopathological diagnosis of subacute sclerosing panencephalitis (SSPE), measles virus antigen was detected by immunocytochemical labelling studies. The formalin fixed, paraffin-embedded thin brain sections labelled with anti-measles antibodies and avidin-biotin complex peroxidase were counterstained with haematoxylin. Only a single area of brain was examined in each patient: cerebellum and parietal lobe. Viral antigen positive reaction was identified within Purkinje cells and extending along dendritic processes in cerebellum, and also in oligodendrocytes of subparietal white matter.     

A novel immunohistochemical detection system using mirror image complementary antibodies (MICA)

AIMS: To describe and illustrate a novel and highly sensitive peroxidase-based immunohistochemical detection system which employs mutually attractive, mirror image complementary antibodies (MICA). METHODS AND RESULTS: To demonstrate the sensitivity of the MICA system alongside the avidin-biotin complex (ABC) method, we selected a range of mouse monoclonal and rabbit polyclonal primary antibodies against antigens that are generally regarded as relatively difficult or impossible to detect on formalin-fixed, paraffin-embedded lymphoid tissue. Compared with the ABC method, the MICA immunodetection method enabled us to dilute primary antibodies up to 200-fold with equivalent or superior immunostaining results and, usually, considerably shortened primary antibody incubation times. CONCLUSIONS: We have described and illustrated a novel immunohistochemical detection system and demonstrated greatly increased sensitivity over the commonly used ABC system. An additional advantage of the MICA system is that it is avidin-free and so avoids non-specific staining due to endogenous tissue biotin.     

Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies.

AIMS--To evaluate the effect of microwave irradiation on the staining quality of a range of commonly used primary antibodies in archival, formalin fixed, paraffin wax embedded material, with emphasis on antibodies that have previously worked successfully only on frozen tissue. METHODS--Immunocytochemistry (streptavidin-biotin complex technique) was performed on histological sections of a range of normal and pathological tissues, after varying treatment with microwave irradiation. The staining quality of each antibody was compared with that achieved without prior treatment of the sections or after enzyme predigestion. RESULTS--Microwave irradiation permitted successful immunostaining with 20 antibodies that stained only frozen tissues before. The staining characteristics of 21 antibodies that were already known to stain formalin fixed, paraffin wax embedded material were improved. Another 39 antibodies did not show enhanced staining with microwave irradiation. The method preserves tissue morphology and produces more consistent staining than that achieved by enzyme predigestion with many antibodies. Microwave irradiation may also allow some primary antibodies to be used at higher working dilutions. The citrate buffer used in this study avoids the necessity of exposure to heavy metal salts. CONCLUSIONS--Microwave antigen retrieval represents an important technical advance within immunocytochemistry that will greatly increase the range of antibodies which can be used to study formalin fixed, paraffin wax embedded tissues.     

Variations in immunohistochemical detection of p53 protein overexpression in cervical carcinomas with different antibodies and methods of detection

Immunocytochemical detection of p53 protein products in paraffin sections is possible with a number of antisera, monoclonal and polyclonal. Few corroborative results amongst different laboratories have been published due to variations in the antibodies, the fixation protocols, and the immunocytochemical methods applied. Antigen unmasking methods employing microwaves or proteolytic enzymes add to the disparity in the percentage of positive cases reported. In this study, paraffin sections of 55 cases of cervical carcinoma were immunostained with monoclonal antibodies p53-DO7 and p53-1801 (a) without section pretreatment, (b) with pronase digestion, and (c) with microwave antigen retrieval in citric acid buffer. Specimens fixed in buffered formalin required antigen unmasking to show positive staining. Pronase digestion caused false-negative immunostaining. Microwave pretreatment with p53-DO7 yielded 100 per cent positivity for p53 proteins but only 7/55 cases with more than 50 per cent positive cells. Monoclonal antibody p53-DO7 detected more positive cases than p53-1801. Immunostaining with antibodies to p53 proteins must be interpreted cautiously as variations in fixation, antibodies, and section pretreatment will significantly affect results.     

A panel of antibodies for the immunostaining of Bouin's fixed bone marrow trephine biopsies.

AIMS: To assess a panel of antibodies on Bouin's fixed bone marrow trephine (BMT) biopsies. These biopsies are widely used in routine diagnosis of various haematological malignancies and may be the sole material available in many centres; however, information regarding the immunostaining of this material is lacking. METHODS: Biopsies were taken from 72 patients presenting with various haematological malignancies (leukaemia, 38; lymphoma, 14; multiple myeloma, 20). A panel of antibodies was assessed on Bouin's fixed BMT biopsies by the alkaline phosphatase-antialkaline phosphatase method. RESULTS: Three B (MB2, LN-2, Ki-B5) and two T cell lineage antibodies (UCHL-1, CD3-r) reliably identified lymphoid cells, while MPO-r, Leu-M1/CD15, and KP-1/CD68 recognised cells from the myeloid or histiocytic/macrophage series. Reed-Sternberg cells were stained by LN-2, Leu-M1, and CD30. Antibodies specific for plasma cells (VS38) and hairy cells (DBA.44) gave a variable pattern of staining. Among the proliferation markers, proliferative cell nuclear antigen but not Ki-67 related antibodies were effective. CONCLUSION: This study presents a panel of antibodies with reactivity not restricted to common fixatives that are also suitable for Bouin's fixed BMT biopsies.     

Detection of toxoplasma membrane antigens transferred from SDS-polyacrylamide gel to nitrocellulose with monoclonal antibody and avidin-biotin, peroxidase anti-peroxidase and immunoperoxidase methods

Toxoplasma (Tp) membrane antigens separated by discontinuous SDS-polyacrylamide gel electrophoresis were electrophoretically transferred to nitrocellulose and detected with avidin-biotin (AB), peroxidase anti-peroxidase complex (PAP) or indirect immunoperoxidase (IIP) methods. In the AB method, the nitrocellulose was treated with biotinylated monoclonal antibodies and avidin-labeled peroxidase. In the PAP method, it was treated with monoclonal antibody, rabbit anti-mouse IgG antibody, goat anti-rabbit IgG antibody and PAP. Of the two, the AB method was the more sensitive and specific for Tp membrane antigen. The PAP method was less sensitive, but did not require chemical manipulation of the antibodies and was convenient and useful for analyzing Tp membrane antigens. The IIP method was more convenient, but had a lower sensitivity than the other methods.     

A rapid immunohistologic method for determining the limit of cancer invasion at the surgical margin of scirrhous carcinoma of the stomach during surgery

The authors used the monoclonal antibody (MAb) S202 as an adjunct for detection of scirrhous gastric carcinoma cells in cytomorphologic preparations of the resected stomach. Twenty-six samples of scirrhous gastric cancer were stained by the avidin-biotin complex technique on fixed frozen sections. MAb S202 was strongly reactive on all sections. In 12 cases, samples obtained from surgical margins were examined by a rapid immunostaining method. In one case, conventional cytologic findings were equivocal, whereas by the rapid immunostaining method single cells were stained darkly, indicating malignancy. Thus, immunohistochemical analysis using MAb S202 should be carried out to determine the limit of invasive carcinoma at the time of surgery.     

Lymphoma immunophenotyping: a new era in paraffin-section immunohistochemistry.

Recent advances in immunohistochemistry have made it possible to investigate lymphomas for the expression of a wide range of antigens in fixed tissues. Epitope retrieval, sensitive detection methods, and the availability of new monoclonal antibodies have all contributed to one's ability to perform detailed immunophenotyping that previously could only be done in cryostat sections or by flow cytometry. Current lymphoma classifications make use of characteristic immunophenotypic profiles that aid in the reproducible diagnosis and subcassification of these neoplasms. The following is a review of the current state of immunophenotyping for lymphoid neoplasms in fixed tissues.