HISTOLOGY

The study of the form of structures seen under the microscope. Also called microscopic anatomy, as opposed to gross anatomy which involves structures that can be observed with the naked eye. The word "histology" came from the Greek "histo-" meaning tissue + "logos", treatise. Histology was a treatise about the tissues of the body and the cells thereof. FIXATION Fixation is a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives is available for use, depending on the type of tissue present and features to be demonstrated. There are five major groups of fixatives, classified according to mechanism of action:

Aldehydes Mercurial Alcohols Oxidizing agents Picrates

DEHYDRATION AND INFILTRATION After the specimen has been fixed, it must have all the water removed if it is to be successfully embedded in paraffin wax. This process of dehydration is the same as that used when sections are taken from water to xylene to enable them to be mounted. Infiltration is the saturation of tissue cavities and cells by a supporting substance which is generally, but not always, the medium in which they are finally embedded. Tissues are infiltrated by immersion in a substance such as a wax, which is fluid when hot and solid when cold. Alternatively, tissues can be infiltrated with a solution of a substance dissolved in a solvent, for example nitrocellulose in alcohol-ether, which solidifies on evaporation of the solvent to provide a firm mass suitable for sectioning. EMBEDDING Embedding is the process by which tissues are surrounded by a medium such as agar, gelatine, or wax which when solidified will provide sufficient external support during sectioning. Tissues are embedded by placing them in a mould filled with molten embedding medium which is then allowed to solidify. At the completion of processing, tissues are held in clean paraffin wax which is free of solvent and particulate matter. SECTIONING Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome. The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it. For light microscopy, a glass knife mounted in a microtome is used to cut 10-micrometerthick tissue sections which are mounted on a glass microscope slide. For transmission electron microscopy, a diamond knife mounted in an ultra microtome is used to cut 50nanometer-thick tissue sections which are mounted on a 3-millimeter-diameter copper grid. Then the mounted sections are treated with the appropriate stain.

STAINING Haematoxylin and Eosin is the most widely used histological stain. Its popularity is based in its ability to demonstrate clearly an enormous number of different tissue structures, its widespread applicability to tissue from different sites and prepared in different ways and its comparative simplicity. Essentially, the Haematoxylin components stain the cell nuclei blue-black, with good intra-nuclear details, whilst the Eosin stains cell cytoplasm and most connective tissue fibres in varying shades and intensities of pink, orange and red. The histological methods are widely used to diagnose diseases such as melanoma, carcinoma and also lupus nephritis.

HISTOLOGICAL DIAGNOSIS OF CARCINOMA OF THE PARATHYROID GLAND

Histological examination IS the study of a tissue specimen by staining it and examining it by Light microscope. Histological sections from all cases of hyperparathyroidism surgically treated at University College Hospital, London, were available, together with clinical records for the period 1950-81. In most cases the initial frozen section examinations had been done by one of us (JFS) as well as the gross tissue descriptions and examination of paraffin sections stained with haematoxylin and eosin and other methods when appropriate. In particular, 19 of the20 cases diagnosed as carcinoma had been examined personally. This appearance is commonly seen on low power examination. It at once arouses the suspicion of carcinoma if the capsule and trabeculae splitting up the tumor tend to be of dense hyaline fibrous tissue similar to that seen in many thymomas. If the dense fibrous tissue is associated with evidence of old hemorrhage or previous surgery, however, it may be a feature of an adenoma; trabeculae of loose fibrous tissue intersecting the tumor may also be found in adenomas.

HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY IN DIAGNOSIS

Despite numerous histochemical, ultra structural and immuno-histochemical studies, differentiation between malignant epithelial pleural mesothelioma and adenocarcinoma of the lung remains extremely difficult. Histochemical and immuno histochemical panel commonly used by pathologists:

MESOTHELIOMA PAS after diastase Cytokeratin (AE1/AE3) CK5/6 Thrombomodulin Calretinin CEA (monoclonal) Leu M1 Ber EP4 Negative, Rarely Positive Positive Positive Positive Positive Negative Negative Negative

LUNG CANCER Positive (adenocarcinoma) Positive Negative Negative Negative Positive Positive Positive

MUCIN HISTOCHEMISTRY: Before the advent of immunohistochemistry, histochemical studies played an important role in the diagnosis of mesothelioma. Mesothelioma produce the acid mucopolysaccharide, hyaluronic acid which can be identified by staining with Alcian blue (pH 2.5) with and without hyaluronidase. Identification of neutral mucin droplets within tumor cells or tubular lumina by periodic acid - Schiff reaction with diastase pretreatment strongly indicates that the tumor is an adenocarcinoma and not mesothelioma.

IMMUNOHISTOCHEMISTRY: Immunodiagnostic of mesothelioma is one based on exclusion. There is no single immuno histochemical marker entirely specific for mesothelioma hence a panel of antibodies is used to establish the diagnosis.

Immunohistochemistry is useful in the following areas: To differentiate between malignant epithelial mesothelioma and adenocarcinoma: A combination of monoclonal antibodies to carcinoembryonic antigen (CEA), Leu-M1, Ber-EP4, MOC31 or B72.3 can distinguish 90% pulmonary adenocarcinoma from pleural mesothelioma. Antimesothelioma antibodies are useful in identification of epithelial mesothelioma. Thrombomodulin is extremely useful and is applicable to formalin fixed, paraffin embedded tissue. To differentiate between malignant epithelial mesothelioma versus reactive mesothelial hyperplasia Epithelial membrane antigen (EMA) - strong and diffuse positive membrane staining and/or Human Milk Fat Globulin-2(HMG2) favors mesothelioma over hyperplasia. EMA may rarely show patchy weak membrane staining of reactive mesothelium. P53 protein is also highly specific for mesothelioma over reactive mesothelial hyperplasia (this is less specific than EMA). Staining for cytokeratin may be useful as it may help in the identification of invasion into deeper structures. To differentiate between malignant sarcomatous mesothelioma from sarcoma, localized fibrous tumor and reactive serosal fibrosis The pancytokeratin marker AE1/AE3 and low molecular weight cytokeratin CAM5.2 are useful in the diagnosis of mesothelioma. Most sarcomas, reactive serosal fibrosis and localized fibrous tumors are cytokeratin negative. Vascular tumors: Mesothelioma do not express vascular markers (CD31, FactorVII) Epithelioid leiomyosarcoma: It may not be possible to differentiate epithelioid leiomyosarcoma from sarcomatoid variant of mesothelioma as smooth muscle differentiation may be present in the latter. Synovial sarcoma: These may be confused with mixed and sarcomatoid variants of mesothelioma. D-PAS mucin or positive immunostaining for adenocarcinoma markers in the epithelial elements are particularly useful. Bcl2 protein is present in almost all cases of synovial sarcoma. It is rarely present in mesothelioma.

To differentiate malignant mesothelioma from lymphoma and thymomas Primary pleural lymphomas are high grade and are differentiated from mesothelioma by using cytokeratin and lymphoid markers. Thymomas arising in pleura may be confused with solid and lymphohistiocytoid pattern of mesothelioma. Immunohistochemistry is of little help in this area.