Immunoperoxidase staining of surface and intracellular immunoglobulin in human neoplastic lymphoid cells.

An immunoperoxidase technique for the optical microscopic detection of cellular immunoglobulin has been used to stain fixed smears of human neoplastic B lymphoid cells. Only four out of 28 cases of chronic lymphatic leukaemia (CLL) showed membrane labelling by this technique. In contrast, when 14 samples from other types of B lymphoproliferative disorder (including hairy cell leukaemia, non-Hodgkin's lymphoma, and prolymphocytic leukaemia) were studied, all samples showed membrane immunoglobulin labelling (confirmed by capping experiments). This discrepancy was attributed to the greater density of surface immunoglobulin present on neoplastic cells in the latter group of disorders compared to CLL. This immunoperoxidase technique is therefore less sensitive than immunofluorescent staining of cells in suspension for the demonstration of neoplastic cell surface immunoglobulin. However, it offers a number of advantages (eg, excellent visualisation of cell morphology, permanence of stained preparations, and applicability to stored samples) which recommend it as the method of choice in certain clinical haematological contexts.     

Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP)

An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.     

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of anti-hapten and anti-carrier antibody-producing cells in lymphoid tissue.

Mice were injected intravenously with 2 mg of a bovine gamma globulin-penicilloyl (BGG-Pen) conjugate. Cells producing specific antibodies against the protein carrier bovine gamma globulin (BGG) and cells producing specific antibodies against the hapten penicilloyl (Pen) could be distinguished simultaneously in the same spleen section using a combined peroxidase (HRP) and alkaline phosphatase (AP) immunocytochemical technique. AP-BGG conjugate was used for detection of anti-BGG-producing cells and HRP-human serum albumin (HSA)-Pen conjugate, prepared by coupling penicillin to HRP-HSA conjugate, was used for detection of anti-Pen-producing cells in the same spleen section. After performing both HRP and AP cytochemistry, cells with a blue-stained cytoplasm represent anti-BGG-producing cells and cells with a red-stained cytoplasm represent anti-Pen-producing cells.     

In situ immunocytochemical staining method for haemopoietic cells grown in vitro

Characterization and enumeration of haemopoietic cells grown in vitro is routinely performed either on unstained cultures or on cultures stained by cytochemical agents. This report describes a novel method for the immunocytochemical analysis of cells grown in plasma clots. The stain is performed in situ by subjecting the whole plasma clot in the culture dish to the staining procedure. The growth of early haemopoietic progenitor cells was monitored in cultures from normal human peripheral blood and proliferating progenitor cells were identified with an anti-human transferrin receptor monoclonal antibody followed by a two-layer immunoperoxidase stain. The number of transferrin receptor positive clusters demonstrable after 5 days of culture was similar to the number of haemopoietic colonies detectable after 12 days of culture.     

Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia

We designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with CD137 (a costimulatory receptor in T cells [4-1BB]) and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. A low dose (approximately 1.5×10(5) cells per kilogram of body weight) of autologous chimeric antigen receptor-modified T cells reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo, with delayed development of the tumor lysis syndrome and with complete remission. Apart from the tumor lysis syndrome, the only other grade 3/4 toxic effect related to chimeric antigen receptor T cells was lymphopenia. Engineered cells persisted at high levels for 6 months in the blood and bone marrow and continued to express the chimeric antigen receptor. A specific immune response was detected in the bone marrow, accompanied by loss of normal B cells and leukemia cells that express CD19. Remission was ongoing 10 months after treatment. Hypogammaglobulinemia was an expected chronic toxic effect.     

Accessory cells in physiological lymphoid tissue from the intestine: an immunohistochemical study

We report a study of the organization of accessory cell populations, in normal mucosal lymphoid tissue from small intestine (8 cases), large intestine (6) and appendix (9) using a panel of monoclonal antibodies and polyclonal antisera in paraffin-embedded tissue. Two populations were identified in dome areas, one positive for acid cysteine proteinase inhibitor and HLA class II (WR18) only and the second positive for S-100 protein, CD68, and WR18 and negative for acid cysteine proteinase inhibitor and factor XIIIa. Superficial colonic mucosal and small intestinal villous tip macrophages stained positively with CD68 and WR18 only, while deeper cryptal and submucosal populations exhibited additional positivity for factor XIIIa, but both populations were negative for acid cysteine proteinase inhibitor and S-100 protein. Germinal centre macrophages were positive for CD68, WR18 and acid cysteine proteinase inhibitor and negative for factor XIIIa, and S-100 protein. T zone dendritic cells included a population which stained positively for S-100 protein, WR18 and were negative for factor XIIIa, CD68 and acid cysteine proteinase inhibitor, an immunophenotype typical of interdigitating dendritic reticulum cells. This distribution of phenotypically identifiable accessory cell subpopulations was apparent at all three sites examined. We suggest that the specialized subpopulations of dendritic cells staining for S-100 protein and for acid cysteine proteinase inhibitor which are restricted to the dome areas, may have a potential role in the transfer of antigen across the epithelium to the germinal centres, while factor XIIIa appears to identify a tissue macrophage population with a potential role in stromal modulation distant from direct antigen challenge.     

Patterns of immunohistochemical staining for proliferating cell nuclear antigen and p53 in benign and neoplastic human endometrium

Signet ring cell differentiation in adenocarcinoma of the prostate is uncommon. In a review of 200 cases of prostatic carcinoma, we identified five cases with this change, all in moderately to poorly differentiated prostatic carcinomas. The signet ring cells in prostatic carcinoma contain an intracytoplasmic lumen, shown on electronmicroscopy to be lined by microvilli. Transition stages were seen from solid to acinar to signet ring cells to mucinous variants. We believe that this change is part of the spectrum of appearances of prostatic carcinoma and should not be regarded as a subtype of specific significance.     

Immunohistologic analysis of lymphoid cells by a rapid double staining method

The development of monoclonal antibodies has allowed characterization of subpopulations of lymphoid cells and of their in situ distribution in tissues. The feasibility of simultaneous localization of two surface antigens was studied by double staining with monoclonal antibodies to B cells, T cell subsets and follicular dendritic reticulum cells (DRC) using the avidin-biotin-complex (ABC) method in sections of human lymphoid tissues (tonsil, lymph node, spleen) and a non-lymphoid tissue, endometrium. Color mixture was avoided when an additional incubation with avidin-biotin-labeled peroxidase and subsequent development in the respective substrate of the first sequence preceded the second staining sequence using the primary antibodies at optimal concentrations. The antigenic profiles were portrayed by contrasting and distinct colors of the reaction products. It was observed that T lymphocytes of the cytotoxic/suppressor and helper/inducer phenotypes were topographically associated with each other and with B cells in B and T cell areas of lymphoid tissues as well as in lymphocytic aggregates in endometria. Subpopulations of these cells were mantled by processes of DRCs in lymphoid follicles. The findings indicate that the double ABC staining method can be used for simultaneous demonstration of two surface antigens in tissue sections.     

A new method for detecting hepatitis B surface antigen in liver by silver staining

Staining of tubular and circular structures within the cisternae of the endoplasmic reticulum of the cytoplasm of liver cells infected with hepatitis B virus was enhanced by the use of 1% aqueous silver proteinate.     

Radioimmunometric assay for hepatitis B surface antigen on the surface of infected cells

It has been difficult to differentiate hepatitis B surface antigen (HBsAg) on the cell surface from intracellular or intramembranous HBsAg that is not exposed to the host immune response. We describe here a radioimmunometric assay for HBsAg on the cell surface using HBsAg producing hepatocellular carcinoma cell lines and fibroblasts transfected with cloned hepatitis B virus DNA as models. The assay is sensitive, specific, simple and takes approximately 1 1/2 h. The procedure may be modified to compare quantitatively cell surface with intracellular HBsAg and to demonstrate other cell associated hepatitis B virus antigens such as HBcAg and HBeAg.