An immunohistochemical characterization of rhesus monkey respiratory secretions using monoclonal antibodies

A series of 12 monoclonal antibodies was made against mucous and serous components of rhesus monkey trachea. These antibodies were used to localize secretory products in the respiratory epithelium by immunohistochemical methods. Mucous cells of the tracheal surface epithelium and submucosal glands were shown by histochemical methods to be a homogeneous population containing periodate-reactive sulfated glycoconjugates. Immunohistochemical staining using the monoclonal antibodies on glycol methacrylate sections serial to those stained histochemically revealed a more antigenically heterogeneous population of secretory cells. Four distinct staining patterns were observed with the 12 monoclonal antibodies. Eight antibodies reacted with most, but not all, mucous and serous cells. Two antibodies recognized subpopulations of secretory cells. One antibody stained tracheal mucous cells with a concentration of reaction product on the luminal border, and one antibody stained only serous cells within the glands. Ten of the 12 antibodies were shown to react in an ELISA with the high molecular weight void volume containing mucous glycoproteins separated on a Sepharose CL-4B gel filtration column. None of the antibodies recognized blood group antigens. We conclude: (1) that production of a large panel of monoclonal antibodies to respiratory tract mucins from primates is feasible, (2) that the monoclonal antibodies will recognize epitopes on biochemically identifiable glycoproteins, and (3) that the intracellular mucous products of tracheal secretory cells exhibit greater heterogeneity than is detectable by conventional histochemistry.     

E-cadherin and calretinin as immunocytochemical markers to differentiate malignant from benign serous effusions

AIM: To investigate the expressions of E-cadherin and calretinin in exfoliated cells of serous effusions and evaluate their values in distinguishing malignant effusions from benign ones. METHODS: Fresh serous effusion specimens were centrifuged and exfoliated cells were collected. Cells were then processed with a standardized procedure, including paraformaldehyde fixation, BSA-PBS solution washing and smears preparation. E-cadherin and calretinin were detected by immunocytochemistry (ICC). RESULTS: In the exfoliated cells of serous effusions, most of carcinoma cells only expressed E-cadherin, and most of mesothelial cells only expressed calretinin, and benign cells (lymphocytes and granulocytes) did not express either of them. For E-cadherin, 85.7% (30/35) of malignant effusions and 8.1% (3/37) of benign fluids were ICC-positive (P<0.001). The sensitivity of E-cadherin ICC in the diagnosis of malignant effusions was 85.7%, specificity 91.9%, and diagnostic rate 88.9%. For calretinin, 94.6% (35/37) of benign effusions and 11.4% (4/35) of malignant effusions were ICC-positive (P<0.001). The sensitivity of calretinin ICC in the diagnosis of benign effusions was 94.6%, specificity 88.6%, and diagnostic rate 91.7%. For diagnosis of benign and malignant effusions by combining E-cadherin ICC and calretinin ICC, the specificities were up to 100% and 97.1%, respectively. CONCLUSION: E-cadherin ICC and calretinin ICC are sensitive and specific in differential diagnosis of benign and malignant serous effusion specimens and specificities are evidently improved when both markers are combined.     

恶性浆膜腔积液脱落细胞中P21 ras蛋白表达状况及其意义

目的:检测浆膜腔积液脱落细胞P21 ras蛋白表达状况,探讨其对恶性浆膜腔积液的诊断价值.方法:将新鲜浆膜腔积液离心后收集脱落细胞,取部分细胞涂片进行常规细胞学诊断,据此将积液分为良性与恶性两组.对余下脱落细胞作"标准化"处理,包括去除红细胞、多聚甲醛固定、调整细胞浓度、制备细胞涂片,然后进行P21 ras蛋白免疫化学染色(SP法).结果:共108例浆膜腔积液,恶性53例,良性55例.39例恶性积液P21 fas蛋白免疫化学染色阳性(73.6%),且多为强阳性或阳性;12例良性积液P21 ras蛋白染色阳性(21.8%),且多为弱阳性或阳性,两组间阳性率(χ2=29.02,P＜0.001)及阳性强度(Uc=6.786,P＜0.001)的差异均有显著性统计学意义.P21 ras蛋白免疫化学染色诊断恶性积液的敏感度为73.6%,特异度为78.2%,诊断符合率达75.9%.结论:浆膜腔积液中脱落细胞P21 ras蛋白免疫化学染色在肿瘤细胞多为阳性,良性细胞多为阴性,对恶性体腔积液的诊断有较大的价值.     

Pulmonary abnormalities in Klippel-Trenaunay syndrome. A histologic, ultrastructural, and immunocytochemical study.

Klippel-Trenaunay (KT) syndrome is a rare, sporadic, congenital vascular disease of unknown etiology. We describe pulmonary findings in an 18-year-old male patient followed up since birth with the KT syndrome. The patient developed pleural and pericardial serous effusions that led to an open lung biopsy. Previous pulmonary findings have been limited to thromboembolic phenomena and pulmonary vein varicosities. On the other hand, reports of lymphatic hyperplasia, aplasia, and hypoplasia in KT have been limited to the extremities. For the first time, we describe lymphatic involvement of the lung in KT. The plexiform hyperplasia of the lymphatic channels with smooth muscle hyperplasia leading to lymphatic obstruction, pleural and pericardial effusions are new findings. The lymphatic nature of the plexiform channels was confirmed by immunohistochemistry. Von Willebrand factor and QD-END/10 monoclonal antibodies either did not react or reacted poorly with lymphatic endothelium, features used to distinguish lymphatic and venous endothelium. Ultrastructurally, the absence of basement membrane continuity further substantiated the lymphatic nature of the channels. From our findings, the lymphatic abnormality in the syndrome appears to be more generalized than previously thought. This entity should be distinguished from lymphangioleiomyomatosis to which it bears a superficial morphologic appearance.     

Detection of metastatic tumour cells in routine bone marrow smears by immuno-alkaline phosphatase labelling with monoclonal antibodies

The present study describes 11 cases (10 carcinomas, one rhabdomyosarcoma) in which immuno-alkaline phosphatase labelling with monoclonal antibodies was used to demonstrate metastatic cells in routine smears of aspirated bone marrow. Carcinoma cells were detected using antibodies against epithelial cytokeratins, milk fat globule membrane antigen and carcinoembryonic antigen, and rhabdomyosarcoma cells with monoclonal anti-desmin. In four of the carcinoma cases it had not been possible to identify malignant cells in routinely stained marrow smears, whilst the case of disseminated rhabdomyosarcoma had initially been diagnosed (and treated) as a case of acute lymphoblastic leukaemia. The anti-cytokeratin antibody was found to be the most valuable of the anti-epithelial reagents used, since it labelled malignant cells in all of the 10 cases of carcinoma and gave the strongest reactions. These results suggest that immunocytochemical labelling should be used in cases of suspected carcinoma whenever conventional examination of marrow smears yields negative results, and furthermore (as illustrated by the case of rhabdomyosarcoma) that the technique is of value for identifying the true nature of poorly differentiated neoplasms in bone marrow.     

Immunocytology in malignant pleural mesothelioma. Expression of tumor markers and distribution of lymphocyte subsets

We studied the reactivity of malignant mesothelioma cells with tumor markers and the phenotypes of lymphocyte subsets in pleural effusions from 14 patients with malignant mesothelioma. For identification of cell surface antigens with monoclonal antibodies, the adhesive slide assay was used. The reaction pattern of mesothelioma cells was found to be CEA negative, Leu M1 negative, EMA positive, BMA-120 positive, My 4 positive, and BA-2 positive. The surface morphology of mesothelioma cells may be of additional help for diagnosis. By these markers, the distinction between mesotheliomas and carcinomas is facilitated. The differentiation of reactive benign mesothelial hyperplasia from malignant mesothelioma by surface marker staining is not yet possible, however. In many effusions in this study, a concomitant T-lymphocytosis was observed with a non-specific increase in the CD4/CD8 ratio, as known for other pleural diseases.     

Characterization of murine monoclonal antibodies against the common acute leukemia antigen (CALLA)

This study presents two murine monoclonal antibodies which react with the Common Acute Lymphoblastic Leukemia Antigen (CALLA). Both antibodies can be used for the diagnosis of common ALL (cALL). Indirect immunofluorescence studies (FACS-analysis) showed that the antibodies react with granulocytes and different human cell lines (Nalm-6, Reh, Raji, CCRF-CEM). The monoclonal antibodies BL-CALLA/1 and BL-CALLA/2 identify a single polypeptide chain of 95 kD. Both antibodies recognize the same or closely related epitope of the CALLA-molecule and are able to modulate in vitro the antigen on the CALLA-positive cell line Reh.     

Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies

Within the hematopoietic system, monoclonal antibodies reactive with antigenic determinants, expressed in a lineage- and stage-restricted fashion, can be used to map myeloid differentiation. We have generated a series of monoclonal antibodies that reacts with myeloid-associated determinants on committed myeloid stem cells and their progeny. Their reactivity with peripheral blood cells was identified by immunofluorescence assays, with bone marrow cells by fluorescence- activated cell sorting, and with committed hematopoietic progenitor cells by both cytotoxic assays and fluorescence-activated cell sorting. Antibody 1G10, which has previously been reported to react with cells of the granulocytic lineage and with a minor subset of mature monocytes, was shown to react with granulocyte-macrophage colony- forming units (CFU-GM). Three antibodies not previously characterized (T5A7, L4F3, L1B2) were shown to react with both granulocytic and monocytic cells and in fluorescence-activated cell sorting studies to detectably stain granulocytic cells at different stages of maturation. These three antibodies also react with CFU-GM, two (L4F3 and L1B2) reacting with all CFU-GM, while T5A7 reacts with only a portion of the day 7 CFU-GM. Antibody L4F3 also reacts with a portion of erythroid burst-forming units (BFU-E). In contrast, the previously reported antibody 5F1, which reacts with monocytic cells, nucleated erythroid cells, and platelets, was shown to react with erythroid colony-forming units (CFU-E). Potential applications of these antibodies to studies of normal and malignant hematopoiesis are discussed.     

Attempts at production of monoclonal antibodies against human breast tumor cells

Mouse monoclonal antibodies have been produced by fusion of x 63 Ag 8 . 653 myeloma cells with spleen cells of mice immunized with single cell suspensions of human mammary carcinoma tissue. These antibodies revealed strong differences in binding to cells from malignant and non malignant mammary tissues. The monoclonal antibodies do not react with usual determinants which are present on normal human blood lymphocytes or in human serum.     

Antigenic phenotype of splenic hairy cells

Hairy cell leukemia, a distinct clinical and morphologic lymphoproliferative disorder, is characterized by the proliferation of mononuclear cells of uncertain derivation. Attempts to identify the cell of origin have used studies either of functional capabilities or of membrane/cytoplasmic antigens. Only a few cases have been studied via monoclonal antibodies. Frozen sections of splenic tissue involved with hairy cell leukemia were studied with a variety of monoclonal antibodies having specificity for differentiation antigens using the avidin-biotinylated peroxidase complex technique. Conventional direct and indirect immunohistochemical study was used for immunoglobulin heavy and light chains. In all but one case, the neoplastic cells expressed monoclonal immunoglobulin. Although T cells were identified in persisting periarteriolar sheaths and occasionally admixed with red blood cells in pseudosinuses, phenotypic expression of intrathymic or peripheral T cell antigens by the proliferating neoplastic cells was not observed. Conversely, expression of B1 and HLA-Dr antigens by splenic hairy cells was documented in all 10 cases. Hairy cell leukemia cells did not express either monocyte antigens (M1 and MO2) or the antigens expressed by early (J5) and intermediate (B2) B cells or plasmacytoid lymphocytes and plasma cells (T10). These immunohistochemical results with monoclonal antibodies provide further evidence that hairy cell leukemia is characterized by a combination of antigens peculiar to mature B lymphocytes.     

Monoclonal antibodies against native ovarian tumor cells: specificity and characterization of the antigen. A preliminary report

Monoclonal antibodies were prepared against native human ovarian carcinoma cells derived from the ascitic fluid of a patient (BH). One antibody, HC7R7, was selected on the basis of its binding to tumor BH cells, to other ovarian tumor cell lines (CAOV-3 and GZL-8), but not to the patient's fibroblasts. One hundred cell smears from ascitic and pleural effusions of tumor-suspected patients were immunostained with HC7R7. All serous ovarian carcinomas and half of the breast carcinomas stained positive with HC7R7; cells from noncancer ovarian aspirates were negative. Mesothelial cells were also stained with HC7R7. A correlation was noted between HC7R7 and OC-125 staining of ovarian tumor cells but not between HC7R7 and c-neu staining of breast tumor paraffin sections. The location of HC7R7-positive material in ovarian tumor cell lines (CAOV-3 and GZL-8) differed from that in the breast tumor cell line (MCF-7). CAOV-3 and GZL-8 showed membrane binding while, in MCF-7, not fully identified intracellular organelles were stained. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots from membrane and cytosol fractions of GZL-8 and MCF-7 showed HC7R7 binding to three protein bands in the membrane fraction and to three other bands in the cytosol, all in the 29- to 68-kDa range. Two of the bands were glycoproteins. The only band that was different in the GZL-8 and MCF-7 cells was a 43-kDa glycoprotein, which was more pronounced in the MCF-7 cells. The possible significance of the new HC7R7 antibodies for detection and survey of ovarian malignancies is discussed.     

Immunocytochemical methods in haematology and oncology

Immunocytochemical investigations of fixed cells are used to enhance diagnostic accuracy in haematology and oncology. The alkaline-phosphatase/anti(alkaline phosphatase) technique, immunoperoxidase and the avidin-biotin technique are the most important methods in immunocytochemistry. Tyramide-enhanced immunostaining is a new powerful technique. Peripheral blood smears and cytospins of mononuclear blood leucocytes have been analysed with this technique and these methods are also useful for analysis of bone marrow, cerebrospinal fluid, serous effusions and fine-needle aspirates. This review describes the results of immunocytochemistry in patients with B- and T-lymphoproliferative disorders, acute leukaemias and Hodgkin's disease. These methods are also useful for the detection of epithelial malignant cells. Immunocytochemistry supports the morphological analysis of cells in these disorders.     

Pleural effusions in the atypical pneumonias

Patients with atypical pneumonias, whether caused by bacterial, fungi, or viruses are associated with pleural effusions. The effusions generally are small and ipsilateral to the parenchymal infiltrate. Usually, the pleural fluid is a serous exudate with a predominance of mononuclear cells. The pleural fluid glucose and, presumably, the pleural fluid pH are not low. The etiologic organism has been isolated from pleural fluid but usually is not necessary to establish the diagnosis. Pleural biopsy is not helpful in diagnosis with the exception of acute coccidioidal pneumonia and effusion. Pleural effusions in the atypical pneumonias are more common than generally appreciated, rarely provide a definitive diagnosis, and resolve spontaneously with treatment of the pneumonia without pleural space manipulation. Furthermore, evidence of pleural effusion is a marker of the atypical pneumonias and directs the clinician along the appropriate diagnostic pathway based on the clinical presentation.     

Antiphospholipid antibodies in a series of 25 cases of lupus without antinuclear antibodies. Comparison with a series of 91 lupus patients with antinuclear antibodies

Twenty-five patients with at least 3 of 1982 ARA criteria of SLE but without the ANA, were compared with 91 patients with 4 or more of the ARA criteria of lupus with positive ANA. The ANA-negative group was characterised by the low incidence of skin involvement, serous effusions and alopecia, and a relatively high incidence of thrombocytopaenia and venous and arterial thrombosis. Three types of antiphospholipid antibodies were looked for: the VDRL, antiprothrombinase and anticardiolipin antibodies by an immuno-enzymatic method. The VDRL was the only antibody which was significantly commoner in the ANA-negative group. Statistical studies showed that the three methods of demonstrating antiphospholipid antibodies detected crossed but not identical specificities. In the ANA-positive group only the antiprothrombinase was associated with a high incidence of venous thrombosis and stroke. In the ANA-negative group, only the anticardiolipin antibodies were associated with a high incidence of arterial or venous thrombosis. Two subgroups may be identified in the group of ANA-negative lupus patients: firstly, those with high anticardiolipin antibody titres with a high incidence of thrombotic and haematological complications, and, secondly, patients with low anticardiolipin antibody levels with a high incidence of cutaneous involvement, serous effusions and Raynaud's phenomenon.     

The Preparation and Properties of Monoclonal Antibodies against Human Granulocyte Membrane Antigens

A series of four monoclonal antibodies has been prepared by hybridization of mouse myeloma cells with spleen cells from mice immunized with human blood granulocytes. The four antibodies all react specifically with granulocytes, failing to stain lymphocytes and other blood cells. Lymphocytic leukaemia cells are not stained, whereas myeloid leukaemia cells give a varied reaction with the antibodies. Studies on marrow, leukaemic cells and cell lines suggest that the four antibodies react with distinct differentiation antigens which are absent from myeloblasts, appear at the promyelocyte, myelocyte or metamyelocyte stage (depending on the antibody in question) and are expressed on all mature granulocytes.     

Identification and characterization of an endothelial, cell-specific antigen with a monoclonal antibody

The purpose of these studies was to use monoclonal antibodies to identify and characterize plasma membrane components unique to the vascular endothelium. Our assumption is that such components may perform some of the specialized functions of the endothelium and, by their identification with antibody probes, we may be able to study further their function and structure. Thus, primary cultures of human umbilical vein endothelium were used to immunize mice whose spleen cells were fused with the mouse myeloma cell NS-1. HEC-1 is a monoclonal antibody derived from such a fusion that appears to react with an antigen located only on endothelial cells. The antigen has been characterized by immunoprecipitation and polyacrylamide gel electrophoresis as a glycoprotein with a mol wt of 180,000 daltons under nonreducing conditions and 90,000 daltons under reducing conditions. Despite a close resemblance to a membrane component shown by others to be a receptor for transferrin, several lines of evidence reported in this paper indicate that this is not the function of the HEC-1 antigen. These data show that monoclonal antibodies can be used to identify and characterize membrane components of the vascular endothelium. Moreover, these probes can be used to inquire about the structure and function of the antigen with which they react.     

Experiments on the production of monoclonal antibodies against lymphocyte antigens in cattle with enzootic cattle leukosis

Hybridomas were produced which secrete antibodies against cell surface antigens of neoplastic bovine lymphocytes. The monoclonal antibodies of three hybridomas react only with leukemic cells but not with other tested cellular and soluble antigens. Four hybridomas secrete antibodies which probably recognize virus-encoded structures on outer membranes of leukemic cells.     

Sensitivity and specificity of monoclonal and polyclonal immunohistochemical staining for West Nile virus in various organs from American crows (<Emphasis Type="Italic">Corvus brachyrhynchos</Emphasis>)

Background  

Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection.     

Detection and initial characterization of synovial lining fragments in synovial fluid

OBJECTIVE: Free fragments of synovium have occasionally been seen in synovial fluid but have not been studied systematically. We wished to establish a method for the reliable detection of these fragments in joint and bursa effusions and begin to characterize them by histochemical and immunohistochemical methods. METHODS: Cell smears, wet drop preparations and cytospins were prepared from 39 consecutive joint and bursa effusions. Paraffin cell blocks were prepared from a subset. Analysis encompassed standard and polarized light microscopy, histochemistry, immunohistochemistry and transmission electron microscopy (EM). Synovial biopsy tissue from one different patient was examined for comparison. RESULTS: Tissue fragments were not seen in Wright-stained cell smears and only rarely in wet drop preparations. In contrast, variously sized fragments with the histological appearance of hyperplastic synovial lining were detected in ethanol-fixed, haematoxylin/eosin-stained cytospins from bursitis and all arthropathies studied [17/24 (71%) of non-inflammatory and 12/15 (80%) of inflammatory specimens]. Immunostaining revealed CD68 expression in a subset of cells in a pattern characteristic of hyperplastic synovial lining. Juxtaposed cells with morphological features of macrophage-like and fibroblast-like synoviocytes were seen by EM. CONCLUSIONS: Synovial lining fragments can be detected in effusions from diverse arthropathies and bursitis. They maintain important properties of the synovial lining and can be analysed by immunohistochemistry. They may afford the opportunity to study a relatively pure preparation of synovial lining cells without the need for cell culture, and to evaluate their possible role in augmenting or perpetuating synovitis or joint damage.     

The CD34 Molecule and Hematopoietic Progenitor Cell Studies - a Challenge in Clinical Medicine

The CD34 molecule, which is expressed at various levels on virtually all human hematopoietic progenitor cells (HPC), is a heavy glycosylated molecule with many different epitopes. A large series of CD34-specific monoclonal antibodies specific for different epitopes has been generated. Some of these antibodies do not react with particular HPC subpopulations. Thus, the antibodies used for HPC enumeration and isolation have to be carefully chosen with respect to what is aimed at.