Department Clin. Sci. Comp. Anim., Veterinary Faculty, Utrecht University, The Netherlands
The Cytological Appearance of the Normal Lymph Node
Although the normal lymph node is seldom aspirated, familiarity with the normal cytological appearance is necessary in order to recognize abnormalities. Mild antigenic stimulation also takes place in the normal lymph node and in principle all of the stages of B- and T-lymphocytes can be found. However, the majority (85-95%) of the cells are small B- and T-lymphocytes. These cells are characterized by little cytoplasm, round nuclei without nucleoli, and often slightly rough chromatin structure. The size of these cells (about 10µm) lies between that of erythrocytes and polymorphonuclear granulocytes. The cytoplasm of lymphocytes is rather fragile and can be found in loose fragments throughout the smear, the so-called lymphoglandular bodies. With the May-Grünwald Giemsa stain they are light blue. Lymphoglandular bodies are characteristic of lymphoid tissue and their presence can be useful in differentiating lymphoid cells from those of an undifferentiated small cell carcinoma. A normal lymph node also contains other developing stages of the lymphoid series but never more than 5-10% of the total number of cells. Other, nonlymphoid cells occurring in a normal lymph node include polymorphonuclear neutrophilic and eosinophilic granulocytes, macrophages, histiocytes, mast cells, erythrocytes, and monocytes. These cells are only present sporadically.
The most frequent cause of the erroneous diagnosis of an enlargement of the mandibular lymph node is the mistaken palpation of a mandibular salivary gland, whether normal or enlarged. In the dog and the cat the mandibular lymph nodes are rostral to the salivary gland and both are in principle readily palpated. Salivary gland cells are much larger than lymphoid cells, contain more cytoplasm, and form acinar (gland-shaped) structures. Lymphocytes and lymphoglandular bodies are absent.
In obese animals it is possible to have the wrong impression that the lymph node is enlarged because it is surrounded by a thick layer of fat. An aspiration biopsy obtains mainly fat. It should be realized, however, that most fat tissue is dissolved in the fixation in alcohol, which is used in most staining methods.
The most frequent cause of a generalized lymphadenopathy is reactive hyperplasia, via which the lymph node reacts to an antigenic stimulus. This can be the result of a viral, bacterial, or parasitic infection, or a reaction to tumor antigens, a foreign body, a skin disorder, or waste products of inflammation somewhere else. Reactive hyperplasia is characterized cytologically by an increase in the number of large blast cells, such as immunoblasts and centroblasts, in relation to the number of small, normal lymphocytes. There are also more mitoses and the number of lymphoplasmacytoid cells (intermediate stage between immunoblast and plasma cell) and plasma cells is increased. Sometimes "Russell's bodies" are seen in the cytoplasm of plasma cells. These are vacuoles filled with immunoglobulins.
Depending on the cause of the stimulation there can also be increased numbers of other types of cells, such as macrophages, polymorphonuclear granulocytes and, especially in skin disorders, eosinophilic granulocytes and mast cells.
The presence of many inflammatory cells in the lymph node is referred to as lymphadenitis. Differentiation into purulent and granulomatous lymphadenitis is made according to the types of inflammatory cells. Purulent lymphadenitis is characterized by the occurrence of many polymorphonuclear granulocytes, usually combined with a light reactive lymphoid population and a few macrophages. The difference from a reactive hyperplasia is sometimes difficult to confirm, but can also be quite clear, as in bacterial lymphadenitis. In the latter case there are many polymorphonuclear granulocytes, necrosis, and sometimes bacteria. Lymphoid cells can even be completely absent. An increase in eosinophilic granulocytes is seen mainly in allergic dermatitis and such parasitic infections as leishmaniasis. If bacteria are present they will be found in granulocytes, while parasites will mainly be found in macrophages.
Granulomatous lymphadenitis is also usually characterized by a slight reactive lymphoid picture and in addition an increase in macrophages, epithelioid cells, and multinucleated giant cells. Epithelioid cells are reticulum cells with an elongated oval nucleus, which is often indented and one end and which has a lightly granular chromatin pattern. Epithelioid cells are often missing cytoplasm in the preparation. Sometimes these cells occur in clusters and can then resemble carcinoma metastases. Granulomatous lymphadenitis is seen in toxoplasmosis, fungal and yeast infections, and certain bacterial infections (e.g., infections with Mycobacterium spp).
A dermatopathic lymphadenopathy is a granulomatous lymphadenitis which occurs with skin disorders in which pruritus, scaling of skin and skin damage are prominent. The cellular picture is characterized by the presence of many brown-black melanin granules and a few eosinophilic granulocytes. Interdigitating cells are also encountered. These are elongated histiocytes with a reticular nucleus and a characteristic indentation of the nucleus.
A complete survey of metastatic malignancies in the lymph node is not worthwhile, for in principle all malignant tumors can metastasize via the lymphatic system. Some types of tumors do metastasize earlier than others to the regional lymph node. Sarcomas generally spread earlier by hematogenous than by lymphogenous routes. Carcinomas, melanomas, and mast cells tumors are often found to metastasize to the lymph node, although this also depends upon the histological subtypes. In principle every cytological preparation from a lymph node in which there are cells that do not belong in a lymph node is suspect for metastatic malignancy.
Primary malignant transformation of the lymph node usually involves cells of the lymphoid system. Such cells as epithelioid cells and histiocytes are seldom involved. In the dog and cat these lymphoid tumors are called malignant lymphoma. They are comparable to non-Hodgkin's lymphoma in man. Since Hodgkin's lymphomas have never been convincingly demonstrated in the dog and cat, the lymphoid tumors in these animals are usually simply called malignant lymphomas.
The cytological appearance of the malignant lymphoma can vary from patient to patient. One assumes that a lymphoid cell in each stage of its development can become malignant, whether by a blockage in further differentiation or by autonomous proliferation of a certain cell type. The cell types which are encountered in malignant lymphoma thus do not differ in appearance from normal lymphoid cells. The cytological differentiation rests on the presence of a monotonous cell population, while in a non-lymphomatous lymph node all different development stages of the lymphoid series are visible.
Various classifications schemes have been developed for non-Hodgkin's lymphomas in man. The Kiel classification (Lennert, 1974) is based entirely on the transformation scheme for normal lymphocytes and is very suitable for cytological purposes (Table 1).
Table 1. Kiel classification according to Lennert, 1974.
If the aspirated cell population consists mainly of characteristic blasts, the diagnosis of malignant lymphoma is not so difficult to make. However, there are also forms of lymphoma in which the tumor cells are difficult to differentiate from mature lymphocytes, especially for the less experienced cytologist. This can be the case, for example, with the lymphocytic and centrocytic lymphomas. Problems can also occur when the lymphoma contains more than one cell type, as in the immunocytic or centroblastic/centrocytic lymphoma. In many of these cases, the cell combination of enlarged lymph node and nonreactive cellular appearance is decisive, especially if the cell population is monomorphic. If in doubtful cases there are a few more plasma cells or other inflammatory cells, more experienced help should be sought or the diagnosis should be confirmed by a surgical biopsy for histological examination.
Morphology of Lymphomas, Classified by the Kiel Classification
This type of lymphoma consists of a monotonic population of small, mature lymphocytes. The cytological appearance is often difficult to differentiate from a nonreactive, normal lymph node. If this picture is found in a definitely enlarged lymph node without any evidence of reactivity, the chance is great that this type of lymphoma is present. If the lymphocytic lymphoma consists of B lymphocytes, which can only be confirmed with certainty by immunotyping, then the nuclei are usually round and have a slightly rough chromatin pattern. This is in contrast to the T-lymphocytic lymphoma, in which the nucleus is slightly indented and has a dense chromatin pattern. Both types of lymphoma exhibit little cell multiplication.
The most important cell type here is the immunocyte, a small lymphoid cell with more cytoplasm than the cells of a lymphocytic lymphoma and a slightly eccentrically placed round nucleus. This cell type has developed a little farther in the direction of the plasma cell. In addition to this cell type a few centrocytes, immunoblasts, and plasma cells can be found. The majority of the cells are, however, small lymphocytes.
The occurrence of plasmacytoma in lymph nodes is extremely rare. In this type of lymphoma mainly atypical plasma cells in diverse stages of development are found.
This lymphoma consists primarily of centrocytes. Centrocytes are small cells with an irregular, sometimes indented nucleus. The cytoplasm is often absent or is very pale. The chromatin pattern is fine and there are usually no visible nucleoli.
As the name indicates, this type of lymphoma consists of both centrocytes and centroblasts. Centroblasts have a large, round nucleus with multiple nucleoli that often lie adjacent to the nuclear membrane. The cytoplasm consists of a thin, dark blue rim. Many mitoses can be found. If the percentage of centroblasts is higher than 30-50%, the lymphoma is called centroblastic.
The most important cell type is the centroblast, but a few centrocytes will often be present. There are two special forms. If there are immunoblasts in addition to centroblasts, the tumor is called a 'polymorphic centroblastic' lymphoma. If there are more than 50% immunoblasts, the tumor is considered to belong to the immunoblastic lymphomas (see below). The other special form is the 'anaplastic centrocytic' lymphoma. Anaplastic centrocytes are large centrocytes with a large, irregularly formed nucleus. The cytoplasm is often more lightly stained than that of the centroblasts. In the dog the centroblastic, polymorphic centroblastic, and anaplastic centrocytic lymphomas the most frequently occurring types of lymphoma.
The lymphoblastic lymphoma is infrequent in the dog. The lymphoblast is a medium-sized, round to oval cell with a thin rim of light to moderately basophilic cytoplasm that is sometimes vacuolated. The nucleus has a fine chromatin pattern with a few small nucleoli. Many mitotic figures can be present. In humans this type of lymphoma sometimes contains 'starry sky macrophages", which are thought to be characteristic of a certain subtype, the so-called 'Burkitt lymphoma'. In the dog, however, this type of macrophage (large, vacuolated macrophages that have phagocytized all kinds of material) are found in various types of lymphomas.
If at least 50% of the cells in a preparation are immunoblasts, a diagnosis of immunoblastic lymphoma is made. Immunoblasts are large cells with a large, round, often eccentrically located nucleus. This nucleus is characterized by one large, centrally located nucleolus. Immunoblasts have a thick margin of blue cytoplasm. In addition to the large immunoblast with a slightly eccentrically located nucleus and much cytoplasm, in the dog there is also a smaller type of immunoblast with a smaller nucleus located centrally in the cell. The common characteristic with the large immunoblast is the large and centrally located nucleolus.
Other types of lymphoma that occur incidentally are mycosis fungoides, histiocytic lymphomas, and multilobated lymphomas. In view of their low frequency of occurrence, they are not discussed in this overview.