Monoclonal antibodies (MT1, MT2, MB1, MB2, MB3) reactive with leukocyte subsets in paraffin-embedded tissue sections.

The absence of reactivity on routinely prepared tissue sections has hampered the use of monoclonal antileukocyte antibodies in diagnostic histopathology. Here we describe five new antibodies reactive with leukocyte subsets in formaldehyde-fixed, paraffin-embedded tissue sections. Antibody MT1 is reactive with mature and immature T cells and not with mature B cells. MT2 is reactive with mature T cells and B cells, but not with immature T cells, activated T cells, and germinal center B cells. Antibody MB1 is reactive with all B cells, with about 50% of mature T cells, and not with immature T cells. MB2 is reactive with all B cells and not with T cells. However, MB2 also stains endothelial cells and several types of epithelial cells. MB3 is reactive with B cells and histiocytes, but not with T cells. The antibodies were tested on a series of lymphomas that were also immunophenotyped with a panel of well-established reagents on frozen tissue sections. The results indicate that the MB and MT antibodies are useful tools in the study of reactive and neoplastic disorders of the lymphoid system.     

Surface markers of lymphocytes in the snake, Spalerosophis diadema. I. Investigation of lymphocyte surface markers.

A specific antiserum was raised in rabbits against thymocytes from snakes, Spalerosophis diadema, and was absorbed repeatedly with snake erythrocytes and kidney cells. In complement-dependent cytotoxicity assays, the absorbed anti-thymocyte serum (ATS) was, at any given dilution, cytotoxic to Sp. diadema thymocytes > peripheral blood lymphocytes (PBL) > spleen cells and could be titrated to a plateau defining a population of about 98% of thymocytes, 80% of PBL and 72% of spleen cells. Antiserum directed against snake immunoglobulins was obtained by injecting rabbits with gamma-globulins separated from snake serum by DEAE-cellulose filtration. the anti-gamma globulin serum was absorbed with snake erythrocytes, and in indirect membrane immunofluorescence stained no thymocytes while reacted with about 15% of PBL and 29% of spleen lymphocytes up to a 1:8 dilution. Fluorescence of positive cells was distributed in spots, patches or caps; cap formation could be inhibited by maintaining the immunofluorescence test at +4 degrees. In each of six separate experiments performed during spring, the percentage of lymphocytes which reacted with anti-snake gamma-globulin serum complemented the percentage of cells recognized by ATS. It was shown, furthermore, that about 3%, 8% and 21% of lymphocytes from thymus, peripheral blood and spleen, respectively, possess a receptor for 2-mercaptoethanol-insensitive antibody-sheep erythrocyte complexes. The results indicate that lymphocyte structural heterogeneity exists in reptiles.     

Comparison of some lymphocyte markers in B-cell chronic lymphocytic leukaemia and systemic lupus erythematosus (the B lymphocyte subset)

Spontaneous mouse red-cell rosette formation (M-rosette), Leu 1 (CD 5) monoclonal antibody binding, ecto-nucleotidase enzyme reactions and mitogen responses of peripheral blood lymphocytes in 10 patients with B-cell chronic lymphocytic leukaemia (CLL), in 8 with systemic lupus erythematosus (SLE) and in 10 controls were studied. The numbers of Leu 1 (CD 5) positive B lymphocytes in CLL and SLE were higher than in controls. These are called "autoregulatory" B lymphocytes, which are thought to be independent of T-cell regulatory effects. A significant increase of mouse rosette forming cells (MRFCs) was found in CLL, while in SLE and in controls their number was low. Compared to the B lymphocytes of SLE patients and controls, those of CLL patients showed diminished responses to pokeweed mitogen stimulation parallel to their decreased level of 5'-nucleotidase. Correlations between these markers and the features of isolated M-rosette positive CLL B lymphocytes are discussed.     

Quantitation and estimation of lymphocyte subsets in tissue sections. Comparison with flow cytometry

A quantitation method for lymphocyte subsets in immunoperoxidase-stained frozen tissue sections was compared with flow cytometry in 23 cases of non-Hodgkin's lymphoma. Close correlations were obtained, demonstrating the accuracy of the technic. Weak intensity of fluorescence and fragility of the tumor cells during the fluorescence-activated cell sorter (FACS) analyses were the most likely explanations for a number of the discrepancies observed. The tissue quantitation method was precise, particularly at low values, where it was better than the FACS. A simpler and faster estimation method employing categories within 10 percentage units was also tested in this study; this method correlated as well with the FACS as the quantitation method and gave the best interobserver correlations.     

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.     

Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. I. A hybridoma secreting antibody against a marker specific for human B lymphocytes

A hybridoma has been isolated from the products of fusion of a myeloma cell line with spleen cells from mice immunized with a human B cell line. After cloning, the hybridoma secretes antibody with the following properties: (i) Human B-lymphoblastoid cell lines react with the antibody while T and null cell lines do not. (ii) The antibody reacts with the majority of leucocytes in the blood of patients with CLL, but with a minority of cells in the blood of patients with AML or ALL of the null or T type. (iii) The antibody reacts with 9-21% of mononuclear cells in normal peripheral blood. The reacting cells are not T cells and overlap extensively with cells identified as B cells by other markers. The antigen identified by this antibody appears to be distinct from known B cell markers, and is put forward as a new B cell marker with diagnostic potential.     

Antigen presentation by B lymphocytes to CD4+ T lymphocytes in vivo: importance for B lymphocyte and T lymphocyte activation.

B lymphocytes, like macrophages and dendritic cells, can present antigen to CD4+ T cells. Antigen presentation by B cells is essential for the generation of an in vivo T cell dependent antibody response, and repeated antigen presentation by B cells to T cells is necessary to induce B cell clonal expansion. Presentation of antigen by resting B cells to unprimed T cells tolerizes T cells, while anti-IgD antibody activates B cells and allows B cell antigen presentation that productively activates T cells. However, activation is not all that is required for B cells to productively present antigen to T cells.     

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.     

Immunocytochemical staining of c-erbB-2 oncogene in fine-needle aspirates of breast carcinoma: A comparison with tissue sections and other breast cancer prognostic factors

The role of fine-needle aspiration (FNA) in breast cancer management is expanding to include prognostic in addition to diagnostic studies. This study was undertaken to compare the immunohistochemical staining of c-erbB-2, a breast cancer prognostic factor, on cytologic FNA smears of breast cancer with that on corresponding formalin-fixed, paraffin-embedded tissue sections of the same tumor and to correlate the c-erbB-2 expression with other known breast cancer prognostic factors. FNA smears (destained, alcoholfixed, Pap-stained direct smears) and corresponding tissue sections (formalin-fixed, paraffin-embedded, unstained sections) from 36 primary breast carcinomas were stained immunohistochemically with c-erbB-2 antibody using an avidin-biotin procedure. Ten tumors (28%) showed strong positive staining for c-erbB-2 on the FNA smear and, of those ten, seven were positive on the corresponding tissue section and three were negative. In several of the cytology and tissue positive cases, staining on the tissue section was significantly weaker than on the FNA smear. Two cases involved treatment with preoperative chemotherapy; in one of those cases, tissue staining was weaker than cytologic staining, and, in the other case, both were negative. Correlation of c-erbB-2 staining with other prognostic factors showed an association between positive c-erbB-2 staining and high nuclear grade. Our results indicate that immunohistochemical staining for c-erbB-2 can be performed on destained FNA smears of breast carcinomas and may be more sensitive on the cytologic specimens than on formalin-fixed, paraffin-embedded tissue sections. Diagn Cytopathol 1994;11:250–254. © 1994 Wiley-Liss, Inc.     

Human blood L lymphocytes in patients with active systemic lupus erythematosus, rheumatoid arthritis and scleroderma: a comparison with T and B cells.

Human blood lymphocytes with high affinity Fc receptors for IgG will bind small aggregates of this immunoglobulin at 4 degrees C. These cells have been named L lymphocytes because of membrane-labile IgG determinants. L cells possess a profile of surface markers and functional characteristics which differ from T and B cells. Immunofluorescence methods have been employed to quantify L lymphocytes in subjects with connective tissue diseases and certain infections, and these values have been compared with those for T and B cells. The mean values of L lymphocytes in groups of patients with systemic lupus erythematosus, rheumatoid arthritis and scleroderma ranged between 14% and 18%; values similar to normals. Groups with acute pneumonia and tuberculosis, however, had significantly increased percentages of L lymphocytes. The absolute number of L cells was decreased in subjects with connective tissue diseases, as was the number of T and B cells. L lymphocytes in those with infections were not significantly decreased. Only L lymphocytes were depleted by immobilized antigen--antibody complexes, another characteristic which distinguishes them from T and B cells.     

Tuberculous pleural effusions: lymphocyte phenotypes in comparison with other lymphocyte-rich effusions

This study investigated whether the analysis of T cell subsets and of activation markers on T cells in pleural fluids can be helpful for diagnostic purposes in tuberculous pleurisy and other lymphocyte-rich pleural effusions. Pleural effusion fluids were obtained from 18 patients with tuberculous pleurisy (TB), 21 with effusions following radiotherapy (RT) for a malignant disease, and 11 with congestive heart failure (CHF). Lymphocyte subsets were analyzed by a battery of monoclonal antibodies using an immunoperoxidase method. The majority of the lymphocytes were CD3-positive T cells (TB, 86 +/- 7% of lymphocytes; RT, 81 +/- 8%; CHF, 84 +/- 12%). The ratios of CD4-positive helper-inducer to CD8-positive suppressor-cytotoxic T cells were higher than those reported for the peripheral blood but not significantly different between the study groups (TB, 3.3 +/- 1.9; RT, 2.8 +/- 1.4; CHF, 2.5 +/- 1.1). The activation marker studies revealed that only a few pleural T cells were positive for CD38, CD25 (interleukin-2 receptor), HLA-DR antigen, and OKT9 (transferrin receptor), the proportion of CD25-positive T cells being higher in TB and in RT than in CHF and the proportion of HLA-DR-positive T cells being higher in TB than in CHF (P less than 0.05). Significant differences were not observed relative to the natural killer-cytotoxic phenotypes staining positive for Leu-7 or for CD16. Thus, we concluded that phenotypic analysis of lymphocytes is of limited diagnostic usefulness to differentiate tuberculous from other nonmalignant effusions.     

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.     

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.     

Spontaneous autorosette-forming cells in man. A marker for a subset population of T lymphocytes?

A subpopulation of human peripheral blood lymphocytes are capable of binding with human (autologous or allogeneic) erythrocytes, forming rosettes. The conditions which lead to autorosette formation are similar to those required for sheep red-cell rosetting. Ageing human erythrocytes are shown to bear less of the determinants involved in the phenomenon than younger ones. Evidence is presented that autorosetting is a T-cell marker. As autorosette-forming cells are very sensitive to the inhibiting effects of ATG they could therefore belong to a T-cell subpopulation.     

Identification of a CD21 receptor-deficient, non-Ig-secreting peripheral B lymphocyte subset in HIV-seropositive drug abusers.

We have studied 61 HIV-seropositive heroin addicts (18 asymptomatic, 20 ARC, and 23 AIDS cases), 26 HIV-seronegative heroin addicts, and 45 healthy blood donors, matching the groups each other for age and sex. We have focused on the phenotypic characteristics of B subpopulations in the peripheral blood of HIV-seropositive and -seronegative drug abusers, paying particular attention to the consistence of the "CD20+" B cell subset, which poorly expresses the CD21 membrane receptor for the C3d and Epstein-Barr virus (EBV) (referred to as "CD20 + CD21-" subset). In healthy blood donors, the ratio CD20 + CD21-/CD20+ x 100 is extremely low (mean +/- SEM = 8.1 +/- 0.9) and rarely exceeds the value of 20. On the contrary, in HIV seropositives, the values are much more dispersed, with higher mean values (mean +/- SEM = 25.8 +/- 1.8) ranging from 50 to 60. An intermediate situation characterizes the class of HIV-seronegative heroin addicts, whose values are slightly higher and more dispersed than that of normal controls (mean +/- SEM = 11.6 +/- 1.3). The extent of the amplification of the CD20 + CD21- subset in HIV-seropositive individuals does not apparently correlate with the progression of the disease and represents an early event in the clinical course of HIV infection. For each subject of the study group, the number of CD20 + CD21- B lymphocytes is not correlated to other early markers of HIV infection, as the T4 lymphocyte number, or total Ig levels in sera. A functional characterization of the CD20 + CD21- B cell subset indicates that, in HIV-seropositive patients, these cells are unable to produce specific and nonspecific immunoglobulins (Ig's), either spontaneously or after pokeweed mitogen stimulation. Furthermore, this cell subset is characterized by poor expression of surface Ig's. The data reported suggest that this cell subset can be regarded as situated at an early level of B cell lineage differentiation.     

Localization of human T lymphocytes in tissue sections by a rosetting technique.

A technique for the formation of sheep erythrocyte (E) rosettes in frozen human tissue sections is reported. The labile nature of the receptor for E rosettes on lymphocytes requires the use of controlled conditions for tissue processing and the reaction with indicator cells. The distribution of E rosettes in sections of normal human thymus, lymph nodes, tonsils, and spleens was comparable to that of the T marker-positive cells identified by immunofluorescence with the specific anti-human T cell serum. There with no overlap with areas positive for 19S EAC and 7S EA rosettes. Erythrocytes treated with a sulfhydryl reagent, 2-aminoethylisothiuronium bromide (AET), and with neuraminidase formed better rosettes in sections than did untreated erythrocytes. E rosettes in tissue sections can determine changes in the distribution of T cells in different lymphoproliferative and infiltrating disorders.     

Distribution of B lymphocyte subsets in normal lymphoid tissue.

Using the ABC avidin-biotin peroxidase techniques and a series of monoclonal antibodies against B cell associated antigens, the anatomical distribution of B lymphocyte subsets was studied in reactive lymph node, tonsil and spleen. Evidence is presented for at least five major phenotypically distinct B cell subsets, each localized to specific compartments of peripheral lymphoid tissue. The possible relationship of these subsets of B lymphocytes to activation, maturation and function in the B cell lineage is discussed.     

Immunoglobulin subunits of murine B lymphocytes: structure and associations with other membrane proteins.

The Ig subunit structure of murine B lymphocytes was studied by employing different radiolabelling techniques in combination with chemical cross-linking. The main membrane structure of IgM was a half molecule that was disulphide-linked to proteins with MW 30,000, 45,000 and 55,000, respectively. Small amounts of mu 2L2, microL disulphide-linked to a protein with MW 50,000, and free microL were also detected. The main IgD structures were half molecules disulphide-linked to two proteins with MW 14,000 and two proteins with MW 16,000. Furthermore, IgD half molecules disulphide-linked to a protein with MW 16,000 and free half molecules could be demonstrated. Labelling with hydrophobic reagents showed that all Ig molecules and the protein with MW 50,000, linked to microL, penetrated the lipid bilayer, whereas the other IgM- and IgD-linked proteins probably did not. Additional proteins which were associated exclusively with IgM were detected by chemical cross-linking. These findings offer new possibilities for the investigation of the function(s) of antigen receptors on B cells.