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Query: UMLS:C0268596 (EMA)
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Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR immunostaining with increasing pH, but only weak immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.
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PMID:Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. 782 75

Microwave oven (mwo) is used to stimulate tissue fixation and to retrieve antigens damaged by fixation. Heavy metal salt solutions, water, and citric acid buffer (cab) have been suggested for this purpose. A serie of tumors treated with cab and phosphate-buffered saline (pbs) with mwo were studied immunohistochemically with 24 antibodies. Controls were treated in the same way, except for microwaving. The antibodies were directed against antigens of the following tumors: breast and prostate carcinoma, carcinoid, lymphoma and melanoma. The results showed that cab enhanced the immunoreactivity of the following antigens: estrogen receptors (AMAC), progesterone receptors (Novocastra), HMB45, vimentin, leukocyte common antigen, PCNA, p53, MIB-1 (Ki-67) and prostatic specific antigen. The antigens that did not improve their immunoreactivity, when compared with the control series were: factor VIII, keratin, Leu 22, L26, neuron-specific enolase, CEA, chromogranin, HBME-1, smooth muscle actin and EMA. Microwaving equally improved protein S100 and desmin either with cab or pbs. The only antigen that improved with pbs was actin. The results with B72.3 and NKI/C3 were poor and not reliable. In conclusion microwaving with cab enhances the immunoreactivity of the antibodies mentioned above leading to an increase in sensibility without loosing specificity.
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PMID:[Antigen retrieval by microwave oven with buffer of citric acid]. 799 28

Seven solitary fibrous tumors (SFTs) of the meninges are presented and their clinicopathologic features are compared with those of 64 fibrous meningiomas (FM). Patients with SFT included 5 females and 2 males age 47 to 73 years. The dura-based tumors involved the parasagittal region (1), tentorium (2), cerebellopontine angle (2), and spinal region (2). One each showed invasion of brain and of a spinal nerve root. Of four SFTs with at least 1-year follow-up, one subtotally resected example recurred. No tumors metastasized. All consisted of spindle cells disposed in fascicles between prominent, eosinophilic bands of collagen. Whorls and storiform cell arrangements were lacking. Mitoses ranged from 1 to 7/10 400 x fields. MIB-1 labeling indices ranged from 1% to 18% (mean 4%). All were PAS negative and showed strong immunoreactivity for vimentin and CD34. Of cases studied, half were estrogen and all were progesterone receptor immunopositive. The majority (72%) of FMs occurred in females and most (72%) were supratentorial. Recurrence was noted in 15%. Mitotic activity varied from 0 to 3 mitoses per 10 400 x fields (mean < 1). MIB-1 labeling indices ranged from 1% to 5% (mean 1.5%). Unlike SFT, FMs were glycogen-containing and variously exhibited a storiform pattern (13 of 20), psammoma body formation (9 of 20), and calcification of collagen (4 of 20). Immunoreactivities included vimentin (100%), focal to patchy EMA (80%), S-100 protein (80%), collagen IV (25%), and patchy, mild-to-moderate CD34 staining (60%). Of cases studied, nearly half were estrogen and all were progesterone receptor staining positive. Meningeal SFTs represent a distinct morphologic entity, the morphologic and immunohistochemical features of which differ from those of FM and suggest a histogenetic relationship to pleural SFT. Although a minority histologically appear to be low grade malignant, our limited experience suggests that they behave in a benign fashion. The classification of mesenchymal tumors affecting the central nervous system must be expanded to include SFT.
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PMID:Solitary fibrous tumor of the meninges: a lesion distinct from fibrous meningioma. A clinicopathologic and immunohistochemical study. 871 77

Paraffin-embedded specimens from 21 patients (mean age 49 years) with malignant melanocytic tumors of the central nervous system were studied. Extraneuronal primary tumors were situated at the trunk (38%), the lower (14%) or upper extremity (10%), and the head/neck region (5%). In 33% no extraneural primary tumor could be detected. The tumor location was frontal (19%), occipital (19%), parietal, spinal, multifocally (14%, respectively), or temporal (5%). Four subtypes were distinguished according to the predominant histological cell type: pleomorphic, epithelioid, spindle- and mixed-cell tumors. 29% contained no melanin, most of them belonging to the epithelioid subtype. The morphology and immunohistochemical reactivity for different antibodies (KL-1, EMA, VIM, HMB-45, NKI-C3, S-100, and MIB-1/Ki-67) were assessed. Positive staining was demonstrated for HMB-45 (in 86% of cases), NKI-C3 (100%), S-100 (95%), vimentin (75%), and KL-1 (33%). No expression of the cytokeratin EMA could be detected. The mean proliferation index measured by MIB-1 immunoreactivity was 21%. The 4 histological subtypes were found to express different antigen patterns. In the analysis of CNS tumors of unknown origin, the panel of antibodies used for diagnosis should include HMB-45 as the most specific marker for malignant melanoma.
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PMID:Malignant melanoma in the CNS, subtyping and immunocytochemistry. 940 2

We report herein on a case of clear cell meningioma originating from the cerebellar tentorium, surgically treated by occipital transtentorial approach (OTT). A 67-year-old woman was admitted to our hospital in September, 1996, complaining of gait disturbance. MRI revealed an isointense mass on T1 and T2 WI, clearly enhanced with gadolinium in the right upper cerebellum originating from the tentorium. A left vertebral angiogram showed tumor stain from the right superior cerebellar artery. The tumor was subtotally removed by OTT on September 24, 1996. Gamma knife radiosurgery was performed for regrowing tumor 6 months after surgery. Histological examination revealed that the tumors were composed of sheets of clear, glycogen-rich cells and lobulated by thin connective tissues. There were no malignant findings, but some tumor cells had infiltrated into the cerebellar cortex. Immunohistochemistry showed that tumor cells were positive for EMA and vimentin, but negative for keratin. MIB-1 staining index was 7.02%. From these findings, this case was diagnosed as a typical clear cell meningioma originating from the cerebellar tentorium. From a review of the literature including our case, clear cell meningioma may be clinicopathologically malignant, so careful follow-up will be necessary.
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PMID:[A case of clear cell meningioma originating from the cerebellar tentorium]. 955 60

Markers of cell proliferation (MIB-1), differentiation (S-100 protein, cytokeratin, transthyretin, GFAP, EMA, CEA), and cell adhesion (CD44) were analyzed immunohistochemically in a biopsy series of 21 chorioid plexus tumors determine their correlation with histological grade and impact on clinical course. The material included 8 papillomas (WHO grade I) and 8 carcinomas (WHO grade III); another 5 tumors were tentatively classified as atypical papilloma. The MIB-1 labeling index of papillomas was 3.7%, while that of carcinomas was 14%. Atypical papillomas (mean: 6%) failed to segregate as a statistically different group. The age and sex of patients, and tumor localization were found not to influence MIB-1 reactivity. High MIB-1 labeling indexes were associated with less favourable postoperative outcome. S-100 protein immunoreactivity was generally reduced in carcinomas, while most tumors were positive for transthyretin and cytokeratin irrespective of their MIB-1 status. Positivity for GFAP and EMA was detected in some tumors of both low- and high grade. CEA was universally negative. The standard isoform of CD44 was only expressed in atypical papillomas and 4 carcinomas showing focal infiltration of adjacent tissues. Western-blot analysis was also carried out in 5 cases to detect CD44. Quantitation of MIB-1 immunohistochemistry may, thus, prove a more readily accessible ancillary method for assessing chorioid plexus neoplasms than analysis of differentiation markers. Our data also suggest that expression of CD44H is instrumental in conferring invasive potential to these tumors and possibly contributes to tumor progression as well.
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PMID:[Prognostic markers in the histopathological diagnosis of tumors of the choroid plexus]. 957 5

We have encountered a series of 8 third ventricular neoplasms with a distinctive chordoid appearance that appear to represent a clinicopathologic entity. The tumors occurred in 7 females and 1 male, ranging in age from 31 to 70 years. In all cases, imaging studies showed a large well-circumscribed third ventricular mass; a cystic component was noted in 2. The tumors consisted of cords and clusters of cohesive, oval-to-polygonal epithelioid cells with abundant eosinophilic cytoplasm, relatively uniform round-to-oval nuclei, and inconspicuous nucleoli. Mitotic activity was absent. The stroma consisted of scant, coarse fibrillar processes, as well as prominent, slightly basophilic, extracellular mucin resembling that in chordomas. Throughout the tumor, and surrounding its well-defined borders, were infiltrates of mature lymphocytes and plasma cells. Russell bodies were prominent in the latter. Adjacent brain tissue showed reactive changes with gliosis and numerous Rosenthal fibers. Immunohistochemically, tumor cells were strongly reactive for GFAP and vimentin, but negative or only weakly staining for EMA. The MIB-1 labeling index was approximately 1%. Ultrastructural examination of 4 cases revealed focal microvilli, scattered "intermediate" junctions, and focal basal lamina formation. Neither desmosomes nor cilia were seen. Total resections were achieved in 2 cases; only subtotal removals were achieved in 6. Subsequent tumor enlargement was noted in 3 of the 6 patients with incomplete resection, and of these, two died at post-operative intervals of 8 months and 3 years. The other patient survives 4 years post-operatively with stable residual disease. Of the 2 patients with total resection, 1 was lost to follow-up; the other, during a brief follow-up period, did well without evidence of recurrence.
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PMID:Third ventricular chordoid glioma: a distinct clinicopathologic entity. 960 Feb 20

We developed the Auto Cyto Fix (ACF) 1000 as an experimental model, which was adapted as a membrane filter method for automatically making smear preparations fixed with 95% ethanol. In this study, immunohistochemistry was applied to the cells smeared on the membrane filter, and effectiveness was examined. Twenty-four effusions and HeLa 229 cells were used. These cell samples were produced by the ACF1000 on the membrane filter and stained by the Papanicolaou method. After observation, these were decolored and stained by immunohistochemical techniques. Antibodies included CEA, EMA, and MIB-1. CEA and EMA were detected by the indirect method, and were colored by DAB, AEC, and new fuchsin. MIB-1 was detected by the immunoperoxidase (LSAB) and the immunofluorescence method. A uniformly stabilized immunoreaction was obtained and was equivalent to or better than that of the conventional technique. In addition, there was no background staining on the membrane filter. Based on these results, the membrane filter preparation produced by the ACF1000 was also effectively applied to immunohistochemical observations.
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PMID:Usefulness of Auto Cyto Fix (membrane filter method) for the application of immunohistochemistry. 1178 90

The etiology and pathogenesis of Hodgkin lymphoma (HL) are not yet known. There are implications of genes involved in programmed cell death (apoptosis), and there have been repeated suggestions of an association with Epstein-Barr virus (EBV). The aim of this study was to investigate the protein expression patterns of key cell cycle-related genes, together with evidence of apoptosis and EBV status, in relation to clinical stage in HLs. A double immunohistochemical and in situ hybridization technique was used to detect the expression of bcl-2, p53, retinoblastoma (Rb), p21, Ki67 (MIB 1), and topoisomerase IIalpha (TopoIIalpha), together with latent membrane protein-1 and EBER for EBV status and TdT-mediated dUTP-FITC nick end-labeling (TUNEL) as a measure of apoptosis, on tissue microarray sections of 62 cases of classic HL (35 NS, 17 MC, 8 LR, and 2 LD). A panel of phenotypic markers was used to facilitate recognition of Hodgkin and Reed-Sternberg (H-RS) cells: CD3, CD20, CD30, CD15, and EMA. The H-RS cells of 62 classic Hodgkin lymphomas were bcl-2-positive in 35 cases (56.45%), p53-positive in 14 (22.58%), and positive for both EBV latent membrane protein-1 and EBER in 37 (59.68%); there was complete concordance of results for EBV by both procedures. No correlation was found between expression of bcl-2, p53, or EBV markers in H-RS cells and clinical stage (P > 0.05). Expression of Rb, Ki67, p21, and TopoIIalpha did, however, show significant differences with clinical stage. Expression of Rb and p21 in CD30-positive H-RS cells decreased with more advanced stage (P < 0.001). In contrast, Ki67 and ToPoIIalpha expression increased with later stage (P < 0.01). No correlation was found between expression of any of these markers in H-RS cells and the subtypes of nodular sclerosis HL, mixed cellularity HL, and LRHL (P > 0.05). TUNEL was found in the nonneoplastic cellular background in all cases and in H-RS cells in only 10 of 62 cases (16.12%) (8 nodular sclerosis HL, 1 mixed cellularity HL, and 1 LRHL). There was a significant correlation between high expression of bcl-2 and a low score by TUNEL (P < 0.05). These data are consistent with the notion that overexpression of bcl-2 may be linked to blockage of apoptosis-mediated death of H-RS cells in classic HL. Abnormal expression of p53-related protein may not play a major role in HL, because it is present in H-RS cells in only a minority of cases. Increased expression of Ki67 and TopoIIalpha by H-RS cells is significantly associated with advanced stage and may indicate aggressive disease. Adverse clinical outcome in HL also is associated with loss of Rb and p21 protein expression, consistent with the possible roles of Rb and p21 in inhibition of the growth of H-RS cells. Within the limitations of the methods used, almost two thirds of cases of HL provide evidence of an association with EBV. The tissue microarray technique is valuable not only for examination of large numbers of cases of a disease by a complex panel of markers but also potentially as a control for staining quality in immunohistochemistry and in situ hybridization.
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PMID:Apoptosis and cell cycle-related genes and proteins in classical Hodgkin lymphoma: application of tissue microarray technique. 1296 46

The correlation between the histological features and clinical outcome remains poor in pediatric intracranial ependymomas. We performed a retrospective study of a group of 31 patients (diagnosed from 1985 to 1995) to assess prognostic implications of the current grading system, of histological and immunohistochemical features, and of ploidy status estimated by flow cytometry. Immunoexpression of a broad spectrum of antigens was evaluated, including MIB-1, topoisomerase-IIalpha, cyclin D1, glial and epithelial proteins (GFAP, EMA, cytokeratins), molecules involved in controlling apoptosis (bcl-2, caspase-3/CPP32), and p53 oncoprotein. Univariate and multivariate statistical analyses were performed to evaluate the influence of each variable on both the progression free survival (PFS) and the overall survival (OS) with at least 7-year follow up. Although we showed a significant correlation between histological grade and prognosis, the current grading system failed in predicting outcome in nearly one third of individual cases. Problems with interpathologist reproducibility were also demonstrated. The extent of surgical resection was the only clinical factor that was associated with survival. Both the PFS and the OS were significantly decreased for the following pathological variables: increased cellularity (>300 nuclei per HPF), mitotic activity of >7 per 10 HPF, increased MIB-1 labeling index (LI), topoisomerase-IIalpha LI, S-phase fraction, and p53 and bcl-2 positivity. Increased cyclin D1 LI was demonstrated to have only a marginally significant impact on PFS. A flow chart modeling was further performed to formulate a scheme for discriminating of prognostic subgroups. Based on that, p53 immunopositivity and/or MIB-1 LI of >5% (after subtotal resection) or MIB-1 LI of >15% (after complete resection) were the strongest indicators of the tumor's aggressive behavior and of a poor prognosis of the disease. Foci of hypercellularity should be specifically looked for in ependymomas for assessing the immunohistochemical studies.
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PMID:Pediatric intracranial ependymomas: prognostic relevance of histological, immunohistochemical, and flow cytometric factors. 1455 80


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