Demonstration of immunoglobulin in malignant lymphomas. Use of an immunoperoxidase technic on frozen sections

We used an immunoperoxidase technic to detect surface and cytoplasmic immunoglobulin in frozen sections of 46 malignant lymphomas. With the immunoperoxidase technic, differential staining with antisera to the various heavy and light chain classes permitted detection and characterization of monotypic immunoglobulin in frozen sections of 15 of 15 nodular lymphomas and 24 of 31 diffuse lymphomas. The immunoperoxidase technic applied to frozen sections is a convenient and reliable method for the detection of immunoglobulin in lymphoid tissues, which can be performed in a pathology laboratory without the need for special equipment.     

Sensitivity and specificity of immunostaining in the diagnosis of mantle zone lymphoma

The diagnosis of mantle zone lymphoma is sometimes difficult to make solely on the basis of morphology because the mantle zone pattern may be present in other disorders such as benign mantle zone hyperplasia, follicular center cell (FCC) lymphomas, and Castleman disease. To distinguish mantle zone lymphoma from the other disorders mentioned previously, the authors performed immunoperoxidase studies on B-5-fixed, paraffin-embedded sections or cryostat sections of lymph nodes from nine patients with a diagnosis of mantle zone lymphoma. The results then were compared with the immunostaining pattern seen in FCC lymphomas and various benign lymphoid hyperplasias. A monoclonal proliferation of mantle zone cells, as shown by staining for immunoglobulin light chains, was noted in the mantle zones and interfollicular areas in all six cases from which cryostat sections were available. The cells in the residual follicular centers uniformly had a polyclonal light chain marking pattern. Two novel monoclonal antibodies (LN-1 and LN-2) that identify FCCs and B-cells in B-5-fixed, paraffin-embedded tissues also were used in this study. Of the six cases in which the monoclonal antibodies could be used, the cells in the residual follicles were uniformly LN-1 positive, LN-2 positive, while the mantle zone and interfollicular cells were almost completely LN-1 negative, LN-2 positive. The data suggest that mantle zone lymphomas are a distinctive neoplasm of monoclonal B-cells of non-FCC origin. The authors conclude that immunostaining is a sensitive technic for identifying a malignant neoplasm of B-cells in the mantle zone and interfollicular areas. In addition, the method is relatively specific and useful for distinguishing mantle zone lymphoma from similar-appearing disorders such as benign mantle zone hyperplasias and certain FCC lymphomas.     

Primary mediastinal non-Hodgkin's lymphomas: a morphologic and immunologic study of 19 cases

Nineteen, primary, non-lymphoblastic, non-Hodgkin's lymphomas were investigated by conventional morphologic studies as well as immunologic studies using the application of a battery of monoclonal antibodies to frozen tissue sections. Seventeen of the lymphomas were diffuse large cell; one was large cell immunoblastic and one was a follicular and diffuse lymphoma of intermediate differentiation. Thirty-seven percent of the lymphomas showed prominent sclerosis, sometimes associated with the superior vena cava syndrome. Six of the cases showed evidence of immunoglobulin production with light chain restriction. Twelve additional cases were shown to be of B-cell lineage by B1/T015 expression but did not show evidence of immunoglobulin production. One case was a T-cell lymphoma of helper phenotype. Ia expression was found in 14 of 18 cases studied.     

Primary lymphomas of the liver. Report of six cases and review of the literature

Six cases of diffuse large cell lymphoma (DLCL) of the liver were studied with immunohistochemistry for common leukocyte antigen (CLA), lysozyme, alpha-1-antitrypsin (AAT), and kappa and lambda light chains on paraffin-embedded tissues. All six cases were positive for CLA. Four of the six cases showed staining for lysozyme and AAT (three focal and one diffuse staining). In three cases, frozen tissue for monoclonal antibodies and glutaraldehyde-fixed tissue for electron microscopic examination were available. Two of these showed B-cell phenotypes with monoclonal antibody studies. Electron microscopic examination on these two B-cell lymphomas showed scant cytoplasm and a paucity of cytoplasmic organelles. The third case did not show definite B- or T-cell surface markers but did show strong Leu-M1 and OKM1 staining. Electron microscopic examination of the tumor cells showed a prominent Golgi apparatus, abundant cytoplasm with numerous cytoplasmic organelles and phagolysosomes. However, DNA hybridization studies on this tumor showed immunoglobulin heavy and kappa light chain gene rearrangements typical of a B-cell lymphoma. All six lymphomas were solitary liver masses without evidence of disease elsewhere. The mean age for the six patients was 56.2 years (four males, two females).     

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.     

Expression of Tac antigen by non-Hodgkin's lymphomas

Anti-Tac is a monoclonal antibody that appears to recognize the interleukin-2 receptor. With the use of a frozen-section immunoperoxidase technic, a large series of non-Hodgkin's lymphomas were investigated for the presence of Tac-antigen on neoplastic cells. Approximately one-fourth of cases expressed the Tac antigen, including 27% of B-lineage lymphomas, 6% of the T-lineage lymphomas, and three of four cases of Ki-1-expressing lymphoma. The B-lineage lymphomas with the highest incidence of Tac antigen expression were the large cell lymphomas, both diffuse and follicular, where about one-half of cases expressed the Tac antigen. All major categories of lymphoma expressed Tac except plasma-cytoma/myeloma, small noncleaved cell (Burkitt's and non-Burkitt's), and lymphoblastic malignancies.     

Immunohistochemical staining of non-Hodgkin's lymphoma with monoclonal antibodies specific for the leucocyte common antigen

Forty cases of non-Hodgkin's lymphoma (NHL) have been stained with monoclonal antibodies (F8-11-13, F-10-89-4 and Dako LC) to the Leucocyte Common Antigen (LC) in both cryostat and paraffin sections with an immunoperoxidase technique. In cryostat sections all B-cell lymphomas (25/25) reacted with F10-89-4 and Dako LC, whilst the majority (23/25) stained with F8-11-13; of the T-cell lymphomas studied, all reacted with F10-89-4 (6/60 and Dako LC (4/4), however 2/6 did not react with F8-11-13. Similar variability in reaction was observed in malignant histiocytosis where 1/3 did not react with F8-11-13 whilst all three reacted with F10-89-4 and Dako LC. In paraffin sections (using the two MCabs F8-11-13 and Dako LC) three of the 25 B-cell lymphomas failed to stain with either F8-11-13 or Dako LC (one lymphocytic lymphoma and two lymphomas showing plasmacytic differentiation). The remainder of the B-cell lymphomas reacted with both antibodies. Six out of 12 T-cell lymphomas did not stain with either F8-11-13 or Dako LC, using our standard immunoperoxidase procedure. Staining with Dako LC was however detected in all cases of T-cell lymphoma when incubation with primary antibody was extended from thirty minutes (standard) to overnight. This study confirmed that LC can be detected in all NHL in cryostat sections, and that in the majority of B-cell NHL the higher molecular weight component of LC was demonstrable using F8-11-13. Difficulty in detecting LC determinants after tissue processing for paraffin sections in a number of cases of NHL, especially those of T-cell type or showing plasmacytic differentiation, suggests that lack of reaction with these antibodies does not always preclude the diagnosis of lymphoma.     

Combination of Hodgkin's disease and diffuse large cell lymphoma: an in situ hybridization study for immunoglobulin light chain messenger RNA

It is not clear whether the rare combination of Hodgkin's disease with non-Hodgkin lymphomas are true composite lymphomas or differentiation stages of one tumour cell clone. We used in situ hybridization and immunohistochemistry for the demonstration of immunoglobulin light chains in order to investigate the relationship between the two lymphoma components. In three cases of nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma the Hodgkin cells, as well as the tumour cells in the diffuse large B-cell lymphoma, showed the same messenger RNA for one light chain. Thus, using in situ hybridization in nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma in a small number of cases a possible genetic relationship between the two components could be shown. In nodular sclerosis combined with diffuse large B-cell lymphoma, in situ hybridization did not support a common clonal origin of both tumour parts. However, a unique clonal derivation cannot be excluded by the techniques applied.     

Reactivity of Leu 1 and T101 monoclonal antibodies with B cell lymphomas (correlations with other immunological markers)

The reactivity of Leu 1/T101 monoclonal antibodies (MoAb) was studied in a series of 69 lymphomas with B cell differentiation and was correlated with other cell markers. A three step immunoperoxidase technique on frozen sections was used to test a panel of 20 MoAb: anti-human Ig (heavy and light chains), To 15 (Pan B cells), Leu 1, T101, Leu 4, Leu 3a, Leu 5, OKT 8, OKT 6, Leu 7, anti-CALLA (IOT 5), Leu 10, anti-HLA-DR, OKM 1 and anti-dendritic reticulum cells (R 4/23). T101/Leu 1 antigen was detected in 24 cases: CLL (11 of 11), diffuse centrocytic lymphomas (four of 11), follicular lymphomas (none of 12), follicular and diffuse lymphomas (seven of 10) and one unclassified low grade lymphoma. This antigen was observed in only one high grade malignant lymphoma. In follicular lymphomas, two results deserve attention: (1) T101+ lymphomas showed most frequently IgM+, IgD+ surface Ig. Inversely, T101 unreactive lymphomas displayed IgM+, IgD+ phenotype. (2) Tp67 antigen (T101, Leu 1) and CALLA (GP 100) were found to be mutually exclusive in these lymphomas. These results suggest that follicular lymphomas could be derived from two distinct germinal center cell populations: IgM+ Ig'D-, Calla+, Leu 1-/T101- and IgM+, IgD+, CALLA-, Leu+/T101+.     

Surface marker studies in chronic lymphocytic leukaemia and non-Hodgkin's lymphoma.

Immunological surface marker techniques were applied in a study of 29 cases of chronic lymphocytic leumaemia and 22 of non-Hodgkin's lymphoma. Surface marker characteristics distinguished 2 subtypes of B lymphocytes. Chronic lymphocytic leukaemia was a monoclonal proliferation of B lymphocytes which produced spontaneous rosettes with mouse erythrocytes and had faintly immunofluorescent surface immunoglobulin. The majority of non-Hodgkin's lymphomas also had their origin from B lymphocytes but in contrast, this subtype did not show receptors for mouse erythrocytes and their surface immunoglobulin was brightly staining and demonstrated "capping". The clonal origin of nodular lymphomas could also be demonstrated on frozen sections stained for surface immunoglobulin. Two cases of true histiocytic lymphoma were identified. The current information available on surface marker characteristics of the leukaemias and lymphomas is reviewed.     

Expression of DR antigens in freshly frozen human tumors

DR antigens are thought to function as differentiation antigens and to restrict immune recognition between T cells and B cells, monocyte/macrophages, Langerhan's cells, and endothelial cells. These antigens are commonly found on tissue culture lines from metastatic melanomas and tumors of lymphocyte derivation but are notably uncommon on cell lines from other malignancies. Using frozen tissue sections, a monoclonal antibody (WI 691-13-17) known to detect an epitope common to all DR alloantigens on the beta (light) chain of DR antigens, and a two-step indirect immunoperoxidase technique, DR antigens were found on all metastatic lesions tested and on many primary tumors and their histogenetic precursors. The technique of using monoclonal antibodies in indirect immunoperoxidase staining of freshly frozen tissue allows individual cells to be assessed for antigen expression and presumably more accurately reflects in vivo antigen expression than results obtained from cells selected by tissue culture methods.     

Expression of the Leu-8 antigen by B-cell lymphomas

The Leu-8 antigen is found on the surface of many hematologic cells, including many T- and B-lymphocytes. With the use of a frozen-section immunoperoxidase technic, 152 B-cell non-Hodgkin's lymphomas were examined for Leu-8 expression. Of these lymphomas, 53% expressed Leu-8. Subclassification of the lymphomas with the use of the International Working Formulation showed that most small lymphocytic, intermediate lymphocytic, and diffuse large cell lymphomas and about half of diffuse small cleaved, diffuse mixed, and follicular lymphomas expressed Leu-8. In contrast, all 17 cases of small noncleaved cell (Burkitt's) lymphoma and 9 of 10 cases of multiple myeloma/plasmacytoma were Leu-8 negative. These results indicate that Leu-8 is expressed on a wide variety of B-cell lymphomas and that differences in Leu-8 expression may be useful in the diagnostic separation of small lymphocytic lymphoma with plasmacytoid features from multiple myeloma/plasmacytoma, and diffuse large cell lymphoma from Burkitt's lymphoma.     

Comparison of histologic and immunologic heterogeneity of non-Hodgkin's lymphomas.

The lymphocyte surface marker phenotype in 11 selected cases of non-Hodgkin's lymphomas was determined with anti-immunoglobulin and mouse monoclonal antibodies against human lymphocyte antigens. A complement-mediated cell cytotoxicity assay on suspensions of the tumor cells was compared with an indirect immunoperoxidase technique on frozen tissue sections. Both methods gave good results in tumors with a uniform cell population, but the frozen section technique was superior in heterogeneous tumors. The six B-cell neoplasms were heterogeneous with respect to expression of surface immunoglobulin and Ia antigens. The five T-cell tumors were morphologically heterogeneous and also highly variable in their expression of different T-cell specific antigens.     

Tissue immunomicroscopic evaluation of monoclonality of B-cell lymphomas: comparison with cell suspension studies

A series of 80 tissues removed from patients having a variety of lymphoproliferative disorders were comparatively studied by cell suspension and cryostat frozen section tissue immunomicroscopic technics. Of 39 cases of non-Hodgkin's lymphomas studied by cell suspension, only 18 had surface immunoglobulins (SIg) markers consistent with monotypia (46%). Conversely, immunohistochemistry showed 18 cases (92%). Among the 18 cases in which there was no correlation between immunohistochemistry and cell suspension studies (46%), a variety of cytologic variants of non-Hodgkin's lymphomas was recognized, including nodular poorly differentiated lymphocytic lymphoma, nodular large cell lymphoma, and a soft tissue plasmacytoma. The lack of correlation between the two technics may be due to several different mechanisms, including the selective enrichment of the suspension by nonneoplastic cell populations resulting in a sampling artifact, the disappearance of endogenous SIg in large or plasmacytoid lymphocytes, and the presence of membrane-bound exogenous polyclonal SIG. Immunohistochemistry represents a reliable, simple technic for establishing monotypia in non-Hodgkin's B-cell lymphomas.     

Expression of vimentin and epithelial membrane antigen in human malignant lymphomas

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in 330 cases of lymphoma (317 non-Hodgkin's and 13 Hodgkin's lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperoxidase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T-cell, 247 B-cell and 29 undetermined lymphomas, and 13 cases of Hodgkin's disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B-cell lymphomas (24.2%). This difference in frequency was statistically significant. Vimentin expression in follicular lymphomas was less frequent than in diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin's disease were positive for vimentin in 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T-cell lymphomas. No positive cases were found among follicular lymphomas. In diffuse non-Hodgkin's lymphomas, EMA was expressed only in mixed and large cell types, but never in smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non-Hodgkin's lymphomas, but anti-vimentin antibody may be used as an adjunct to the diagnosis of R-S cells in Hodgkin's disease.     

Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies

Detection of peritubular capillary (PTC) C4d deposition in tissue sections of renal allograft biopsies became an important aid in the diagnosis of antibody-mediated rejection. Pathologists in many major transplant centers now routinely stain renal allograft biopsies for C4d. Currently, there are 3 commercially available antibodies. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections. A polyclonal antibody is used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system. The goal of our study was to compare these antibodies and methodologies in our renal allograft biopsy material. Twenty renal allograft biopsies with diffuse or focal PTC C4d staining, using immunofluorescence methods on frozen sections, were selected for this study. These biopsies were tested with the 3 commercially available anti-C4d antibodies (Biogenesis, Brentwood, Calif, cat no. 222-8004; Quidel Corporation, Santa Clara, Calif, cat no. A213; and ALPCO Diagnostic, Windham, NH, cat no. 004-BI-RC4D). Both monoclonal antibodies (Biogenesis and Quidel) were tested with a 3- and a 2-step indirect IF method on frozen sections. The polyclonal antibody (ALPCO) was applied to formalin-fixed paraffin sections using immunoperoxidase methodology. In selected cases, the polyclonal antibody was tested on frozen sections with a 3-step indirect IF method. To exclude possible false-negative staining with the IF method, we selected 10 additional biopsies that showed PTC margination of inflammatory cells, but were C4d-negative or only focally positive, and tested them with the ALPCO antibody on paraffin sections. We have found that all methodologies and antibodies tested provided adequate results with only minor differences between them. Perhaps the most sensitive method is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. We prefer the 2-step indirect IF method with the Quidel monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost. The advantages and disadvantages of the individual methodologies are discussed.     

Cell proliferation in non-Hodgkin''s lymphomas. Digital image analysis of Ki-67 antibody staining.

Ki-67 is a monoclonal antibody to a nuclear antigen present in cycling human cells but not in resting cells. The authors have performed immunoperoxidase on non-Hodgkin's lymphomas using Ki-67 antibody in order to correlate proliferation rates with tumor grade and type, and compare Ki-67 staining with S-phase content as determined by flow cytometry. Ki-67 staining of 109 sections was quantitated using a digital image analysis system (CAS 100). There was a significant difference among mean overall Ki-67 staining values in Working Formulation low (13.7%), intermediate (42.6%), and high grade tumors (57.9%, P less than 0.00001). The level of significance improved when a revised grading system was formulated based on proliferative activity, with the inclusion of diffuse large cell lymphomas in the high grade category. Within nodular and a few diffuse lymphomas, there were well-defined proliferation centers in which Ki-67 staining showed no correlation with grade. Flow cytometric DNA determination was performed on 74 specimens, and there was a positive correlation between Ki-67 positivity and S phase content (r = 0.66). It is concluded that Ki-67 staining of tissue sections is an alternative to flow cytometric quantitation of cell cycle activity in lymphomas, and provides the advantage of revealing histologic patterns of proliferation. By including G1 phase cells, Ki-67 staining allows a more complete determination of total cell cycle activity in lymphomas.     

Silver enhancement of polymerised diaminobenzidine: increased sensitivity for immunoperoxidase staining

Unambiguous identification of lymphocytes is sometimes difficult because of weak immunostaining of the cell membrane immunoglobulins. A simple method of intensifying the diaminobenzidine (DAB) peroxidase reaction was therefore devised. Paraffin wax sections of formalin fixed tonsils and lymphomas were digested with trypsin and immunostained for kappa and lambda light immunoglobulin chains and CD3 antigen by various peroxidase linked detection systems. After reaction with hydrogen peroxide and DAB the sections were immersed in methenamine silver solution at 60 degrees C for three to seven minutes. The light brown stain on the cell membranes of the mantle zone lymphocytes became dark brown and the stronger stain of the plasma cells became black. Mantle zone B lymphocytes and CD3 positive T lymphocytes were precisely outlined even at low magnification and the lymphomas were easily classified as monoclonal or polyclonal. At high magnification, staining was clearer than with the immunogold-silver stain. Cryostat and paraffin wax sections of other tissues immunostained for various antigens showed similar intensification. Silver methenamine provides an easy means of increasing the sensitivity and visual impact of an immunoperoxidase/DAB reaction in any preparation.     

Demonstration of distinct antigenic profiles of small B-cell lymphomas by paraffin section immunohistochemistry.

The immunoperoxidase technique was used with antibodies against B-cell-associated antigens, including CD20, CD79a, CD10, CD23, CD43, cyclin D1, bcl-2, and kappa and lambda immunoglobulin light chains on formalin-fixed and B5-fixed tissue sections of follicular, small lymphocytic, mantle cell, and marginal zone lymphomas. Results obtained with paraffin section immunohistochemistry for CD20, CD10, CD23, and kappa and lambda light chains were compared with results obtained with flow cytometry or frozen section immunohistochemistry. Cells in all of the lymphoma types were positive for CD20 and CD79a. The antigenic profiles of the B-cell lymphomas demonstrated in paraffin sections were lymphoma type distinctive. Intrafollicular lymphocytes in follicular lymphomas were positive for CD10 and bcl-2. Small lymphocytic lymphomas expressed CD43 and CD23 and were negative for CD10 and cyclin D1. Mantle cell lymphomas characteristically expressed CD43 and cyclin D1 and were negative for CD23 and CD10. Marginal zone lymphomas were negative for CD23, CD10, and cyclin D1. All of the antibodies performed better in B5-fixed tissues, but formalin-fixed tissue immunophenotypes were always similar to those obtained on the B5-fixed tissue. These results were possible using well-fixed tissue, various antigen retrieval strategies, paraffin section reactive primary antibodies, and sensitive detection systems. Paraffin section immunohistochemistry on sections of routinely fixed tissue can be used similarly to flow cytometry and frozen section immunohistochemistry when classifying the lymphomas of small B lymphocytes.     

Immunologic phenotype in 30 patients with diffuse large-cell lymphoma

Frozen sections of 30 diffuse large-cell ("histiocytic") lymphomas that had arisen in both nodal and extranodal sites were stained with immunoglobulin light-chain and heavy-chain reagents, with nonoclonal antibodies to THAT HAD ARISEN IN BOTH NODAL AND EXTRANODAL SITES WERE STAINED WITH IMMUNOGLOBULIN LIGHT-CHAIN AND HEAVY-CHAIN REAGENTS, WITH MONOCLONAL ANTIBODIES TO T and B-cell antigens, and with an esterase marker for macrophages. Fourteen lymphomas expressed immunoglobulin light chains and were shown to be monoclonal; F(ab')2 fragments were sometimes necessary to demonstrate their monoclonal nature. Mu (IgM) was the most frequently expressed heavy chain, but in many patients other heavy chains were found. None of the lymphomas stained with T-cell antibodies or the esterase; 15 did not stain for immunoglobulin, but 13 of these 15 did express Ia antigen. These immunologic markers identified eight different phenotypes. Retrospective clinical analysis of the patients suggested that those who were immunoglobulin-positive had more advanced disease and shorter survival, but confirmation of the clinical relevance of immunologic phenotype will require prospective studies.