[Malignant lymphomas expressing an epithelial membrane antigen. Value in the immunohistochemical diagnosis of tumors].
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Publications and source records attributed to H Stein.
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Epithelial membrane antigen (EMA) is generally assumed to be expressed only on epithelial cells. However, EMA was also found on reactive and neoplastic plasma cells, on some non-Hodgkin's lymphomas (particularly cases of T-cell lymphoma and "malignant histiocytosis"), and on Reed-Sternberg cells in cases of lymphocyte-predominant Hodgkin's disease. EMA was also induced on normal blood lymphocytes by exposure to human T-lymphotropic virus type II or phytohaemagglutinin. The specificity of these aberrant reactions was confirmed by the use of three different monoclonal anti-EMA antibodies (E29, HMFG2, and LICR.LON/M8), and by showing that EMA in lymphoid tissue is similar in molecular weight to EMA in human milk. These results indicate that additional anti-epithelial antibodies (eg, anti-cytokeratins) should be used in conjunction with anti-EMA for tumour diagnosis.
The immunopathological features of lymphocytic non-Hodgkin lymphomas (NHL), follicular centre cell tumours and "large-cell" NHL are described with particular emphasis on in situ evaluation of cryostat sections using monoclonal antibodies. The immunological phenotypes of tumour cells and the pattern of "reactive" cellular infiltrates with characteristic differences for the various subtypes are summarized. Immunological membrane features of the following lymphocytic NHL of B lymphocyte origin are discussed in detail: B-CLL (most common phenotype of the lymphoma cells: VIB-C5, To-15, Ia, Leu-1), lymphoplasmocytoid NHL (To-15, Ia), B-prolymphocytic leukaemia (VIB-C5, To15, Ia, Leu (+/-), FMC-7(+/-] and hairy-cell leukaemia (HCL: To-15, Ia, FMC-7). NHL of T-lymphocytes detailed in respect of mycosis fungoides and Sézary syndrome (OKT-3, OKT-4, OKT-11), to subtypes of T-CLL with particular emphasis on the "azurophilic" type, to T-zone NHL, and to adult T-cell leukaemia/lymphoma (OKT-3, OKT-4, OKT-11, anti-tac), all of them with the usual immunological phenotype of T-helper (TH) lymphocytes. NHL with a predominance of T-suppressor (TS) lymphocytes include the immunoblastic lymphadenopathy like T-cell lymphoma (OKT-3, OKT-8, OKT-11) and some lymphoepitheloid (Lennert) lymphomas. Immunological features of follicular centre cell tumours are discussed with emphasis on similarities and differences with the normal germinal centre. Tumour cells usually show the following membrane phenotype: centroblastic/centrocytic (cb/cc: To-15, VIL-Al, Ia; meshwork of non-neoplastic R4/23+, To-5 + DRC), centrocytic (VIB-C5, To-15, Leu-1, Ia; R4/23 and To-5 meshwork), centroblastic (cb, To-5, To-15, Ia, VIL-Al +/-). "Large-cell" NHL include the last entity (cb), immunoblastic (B-ib, T-ib) and lymphoblastic (lb) NHL and are discussed in respect to B-ib (Ia, To-15 +/-, VIL-Al +/-), T-lb (WT-1, OKT-6 +/-, OKT-11 +/-), pre-B-lb (VIB-C5, Ia, TdT, VIL-Al +/-) and Burkitt-type NHL (VIB-C5, VIL-Al, Ia, sIg). "Reactive" cellular infiltrates may account for a large fraction of the cellular content of NHL (e.g. the mean TH and TS content amount to about 40% of that seen in normal lymph nodes). In comparison to other NHL B-CLL and cb/cc showed the highest, HCL the lowest number of TH X TS exhibited less typical alterations.(ABSTRACT TRUNCATED AT 400 WORDS)
Conventional fixation and paraffin embedding of human tissue destroys most of the antigens detected with currently available antibodies, while cryostat sections give poor morphological detail and require freezer space for storage. However, when tissue was freeze-dried before being embedded in paraffin S then stained for a wide range of antigens recognised by monoclonal antibodies (including T-cell and B-cell antigens), morphological preservation was clearly better, and the labelling reactions were of equal (or greater) intensity, than with cryostat sections. As the number of diagnostically important monoclonal antibodies continues to increase the use of freeze-dried paraffin sections should facilitate the introduction of immunohistological techniques for both routine histopathological diagnostic work and immunological research.
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Follicular dendritic cells (FDC) are specialized cells found only within lymphoid follicles. They bind immune complexes and play a role in the presentation of antigen to follicular B cells and in the generation of B cell memory. In the present report the isolation of FDC from human tonsils and adenoids is described. These isolated cells have an unusual spherical arrangement and enclose lymphocytes within extensions of their membranes. Their ultrastructural features are similar to those observed in situ. The reactivity of isolated FDC with a number of monoclonal antibodies was analyzed by immunofluorescence and by immunostaining (at the electron microscopic level) with colloidal gold. In keeping with the results of previous investigations on tissue sections IgM, IgG and IgA (but not IgD) can be detected on the surface of isolated FDC, as can C3b receptors and the FDC-associated antigen detected by monoclonal antibody R4/23. The immunoglobulins associated with FDC are mostly embedded in an electron-dense material. The majority of the lymphoid cells enclosed within the membrane extensions of FDC are of B cell type. These results suggest that isolated FDC may be suitable for further in vitro investigation of their role in the humoral immune response.
Tracheal transplants were performed on inbred strains of rats. Monoclonal antibodies were used in combination with the biotin-avidin technique for demonstrating donor-specific RT1 Ac transplantation antigen. Transplantation antigens were predominantly contained in the tracheal mucosal cells. No donor-specific antigens could be found in the mucosa of transplants which survived orthotopic tracheal transplantation for longer than 210 days. This suggests that the mucosa of the transplant is rejected and replaced by a mucosa from the recipient.
The antigenic properties of epidermal Langerhans cells (LC) were determined and compared with those of non-lymphoid dendritic dermal cells (DDC), interdigitating reticulum cells (IRC), dendritic reticulum cells (DRC), and histiocytic reticulum cells (HRC) by examination of serial and double immunoenzymatic and -fluorescence stained frozen sections of skin and lymph node biopsies. All of these cell types expressed leucocyte common antigen. LC, DDC, and IRC demonstrated similar antigenic phenotypes (HLA-DR+, Leu3+, OKT6+/-, anti-C3 receptor-, R4/23-, Ig-complex-, MO2-), whereas the antigenic properties on DRC (HLA-DR-, Leu3-, OKT6-, anti-C3 receptor+, R4/23+, Ig-complex+, MO2-) and HRC (HLA-DR+/-, Leu3-, OKT6-, anti-C3 receptor+ R4/23-, Ig-complex+, MO2+) were markedly different. These data suggest that LC, DDC, and IRC are closely interrelated cell types, and support the concept that DRC and HRC are unique cell types which do not appear to be related to LC, DDC, or IRC. The lack of labelling of LC with monoclonal anti-C3b receptor antibody, and polyclonal antiserum recognizing C3b, C3bi, and C3d receptors strongly indicate that the EAC-rosetting of LC previously described is not due to the presence of C3 receptors on these cells. Alternatively, LC may express C3 receptor molecules different from those previously identified (C3b, C3bi, and C3d).
In a prospective study of 376 orthopaedic patients, the relative contribution of host factors and patient-care variables to the risk of postoperative wound infection was evaluated. Host factors studied were age, sex, ethnic origin and diagnosis. The number of operations, the insertion of an open drain, the use of prophylactic antibiotics and the length of the operation were the patient-care variables studied. Of the risk factors identified, the performance of more than one operation during an admission had the highest risk coefficient, followed by the presence of an open drain, internal fixation of a fracture, and spine fusion. Within the group of operations for internal fixation, those for fractures of the femur had the highest risk of infection. In spine fusions those operations lasting 5 or more hours were associated with a high risk of infection. The length of stay of infected patients was on average 17.9 days longer than that of their individually-matched non-infected controls.
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The value of immunohistological labeling with a panel of monoclonal antibodies in the diagnosis of routinely processed surgical biopsies has been assessed. The cases examined consisted of an unselected series of tumor biopsies referred during a 12-month period because of doubt as to the nature of the neoplasm and are representative of the type of diagnostic problem regularly encountered in routine surgical pathology. In 30 of the 38 cases studied, reactivity with monoclonal antileukocyte antibody (and nonreactivity with monoclonal antiepithelial antibodies) indicated that the tumor was a lymphoma. Seven of the remaining eight cases gave the reverse reaction pattern and therefore were classified as carcinomas, while one biopsy was unreactive with all antibodies. Review of the clinical details in each case showed that the clinical management in several instances was influenced by establishing the correct diagnosis and it therefore is suggested that immunohistologic examination should be used more widely in the study of tumors that give rise to diagnostic difficulty.
Skin biopsies from nine patients with a histological and/or clinical diagnosis of cutaneous lymphocytoma, lymphoplasia or Jessner's lymphocytic infiltrate were examined by immunoenzymatic labelling with a panel of monoclonal antibodies against lymphocytes and accessory cells. Similar cellular constituents were demonstrated in the biopsies from three patients with lymphocytoma, two with lymphoplasia and two with atypical lymphocytes, but a protracted benign clinical course. The infiltrates from these patients consisted of T cells, Langerhans cells, related HLA-DR positive dendritic dermal cells and clusters of polyclonal B cells. In four patients, the B cell clusters contained B cell accessory follicular dendritic cells, and thereby closely resembled the B cell follicles seen in lymphoid organs. The T cells were predominantly T helper/inducer cells and in all patients the T cells expressed HLA-DR. One patient diagnosed as lymphocytoma cutis differed from the other patients by having no detectable B cells. One patient with Jessner's lymphocytic infiltrate differed from the other patients by having a marked relative predominance of T suppressor/cytotoxic cells. These data suggest that cutaneous lymphocytoma and lymphoplasia are basically similar disorders which may be considered to be exaggerated immune responses, whereas Jessner's lymphocytic infiltrate may be a separate entity. Immunological analysis may assist in establishing a definite diagnosis in cases of lymphocytoma or lymphoplasia with atypical cytological features.
This paper describes the use of a recently developed immuno-alkaline phosphatase method (the 'APAAP' technique) for labelling frozen sections of undecalcified bone marrow biopsies with monoclonal antibodies, including reagents reactive with T cells and their subsets, B cells, glycophorin, HLA-DR antigen, common ALL antigen, epithelial cells and megakaryocytes. Use of an immuno-alkaline phosphatase technique avoids problems due to endogenous enzyme activity encountered when staining bone marrow by immunoperoxidase procedures. Immunohistological labelling of frozen trephine biopsies is of particular value when it is impossible to aspirate marrow particles and for identifying cells which do not readily enter suspension (e.g. dendritic reticulum cells or stromal cells). Details are given of cases in which immunohistological analysis was used for the phenotyping of acute leukaemias, for the differential diagnosis of intramedullary T and B cell proliferations, and for identifying bone marrow metastases.
Cryostat sections of lymphoid tissue from 44 cases of Hodgkin's disease were analysed by immunoperoxidase staining using a panel of monoclonal antibodies which included reagents reactive with T cells and their subsets, B cells, HLA-DR, Ig, dendritic reticulum cells and C3b receptor. A wide spectrum of immunohistological patterns was observed ranging from cases in which T cells were numerous (B cells being absent or present in only small numbers) to cases in which very prominent B cell follicles were present. These follicles contained a meshwork of dendritic reticulum cells and were composed of polyclonal B cells (as assessed by light chain expression). T cells were present in small numbers within these B cell follicles, often clustered in a thin rim around individual Reed-Sternberg and Hodgkin's cells. All B cell-rich cases were examples of lymphocyte predominant Hodgkin's disease. Assessment of the T cell helper/suppressor ratios was hindered by the fact that both anti-helper antibodies (OKT4 and anti-Leu 3a) reacted with macrophages. However the majority of cases appeared to contain a normal excess of T helper cells. HLA-DR was strongly expressed in T cell rich areas, on Reed-Sternberg and Hodgkin's cells, on vascular endothelium and on numerous infiltrating cells in the fibrous tissue areas in cases of nodular sclerosing disease. Reed-Sternberg and Hodgkin's cells were not labelled by either anti-fibronectin or by antibodies reactive with dendritic reticulum cells (anti-C3b receptor and antibody R4/23).
In a previous study employing conventional immunological marker analysis we found that 17% of high grade malignant lymphomas were devoid of cytoplasmic and membrane immunoglobulin and also sheep erythrocyte receptors. Cryostat sections from 24 of these cases (four of low grade and 20 of high grade malignancy) were stained with a panel of 30 monoclonal antibodies and six polyclonal antisera using a sensitive immunoperoxidase method. All tumours expressed the leucocyte common antigen (detected by monoclonal antibody 2D1) and all lacked epithelial cytokeratin (monoclonal antibody LE61), confirming their haematopoietic origin. All but one of the lymphomas expressed antigens characteristic of either B cells (17 cases) or T cells (six cases), while one case (morphologically a centroblastic lymphoma) had an unusual dual phenotype in which strong staining for T6 (marker of immature T cells) was associated with expression of the pan B lymphocyte antigens detectable with To15, anti-B1, anti-Leu12. This case was therefore classified as a B cell lymphoma showing aberrant expression of the T6 antigen. The pan B cell antibodies (To15, anti-B1, anti-Leu12) all appeared highly specific and sensitive, but the simultaneous use of all three monoclonal antibodies was necessary to detect the B cell nature in each of the 18 lymphomas. A wider panel of monoclonal antibodies was required to detect T lymphomas since these often disclosed atypical and restricted phenotypes. To15 and UCHT1 were the most reliable antibodies for the detection of B and T cell neoplasms, respectively. We conclude that most, if not all, "non-B, non-T" lymphomas are of either B or T lymphocyte origin and that monoclonal antibodies provide indispensable tools in their classification and diagnosis.
A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").
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The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is restricted in its expression to certain phases of the cell cycle. All experiments consistently showed that the Ki-67 nuclear antigen is present in S, G2, and M phase, but is absent in G0. However, the results concerning Ki-67 antigen expression in G1 phase varied: cells passing the early events of mitogen triggered transition from G0 to G1, i.e., G1T and first G1A, lacked the Ki-67 nuclear antigen, whereas G1 cells after mitosis were constantly Ki-67-positive. This result suggests that after mitosis cells might not follow the same metabolic pathways as G0 cells do when entering G1 for the first time. Therefore, we suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle. Because our data show that the Ki-67 nuclear antigen is present throughout the cell cycle, immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.